4 MARCH 2022, VOL 375, ISSUE 6584 Science

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Quantum control of chemistry by magnetic fields pp. 975 & 1006

Soil microbes restoring degraded land p. 990

Comprehensive transcriptomic cell atlas of the fruit fly p. 991

$15 4 MARCH 2022 SPECIAL ISSUE science.org

THE MISSING PHYSICISTS How physics excludes Black researchers p. 950

Apply for our exciting research Prize!

$25, 000 Grand Prize! Get published in Science! The Science-PINS Prize is a highly competitive international prize that honors scientists for their excellent contributions to neuromodulation research. For purposes of the Prize, neuromodulation is any form of alteration of nerve activity through the delivery of physical (electrical, magnetic, or optical) stimulation to targeted sites of the nervous system with impications for translational medicine.

For full details, judging criteria and eligibility requirements, visit:

www.science.org/pins

Submissions Open: 15 December 2021

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CONTENTS

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SPECIAL SECTION

Black physicists INTRODUCTION

950 Can U.S. physics overcome its record of exclusion? Data show white institutions falling short and Black colleges leading the way By J. Mervis FEATURES

952 The toll of white privilege How the dominant culture in physics has discouraged diversity By J. Mervis

956 Fix the system, not the students Change requires building bridges, removing barriers By J. Mervis

960 Black colleges can’t do it all At historically Black colleges and universities, tight funding threatens an extraordinary record of nurturing Black physicists By J. Mervis

970

NEWS IN BRIEF

Close scrutiny of earliest cases and samples from market suggests virus crossed over from animals sold there

940 News at a glance

By J. Cohen

IN DEPTH

948 U.N. panel warns of warming’s toll and an ‘adaptation gap’

942 War in Ukraine poses stark choices for scientists CREDITS: (PHOTO) CYRIL RUOSO/MINDEN PICTURES; (ILLUSTRATION) C. SMITH/SCIENCE

946 Studies bolster pandemic origin in Wuhan animal market

As Ukrainian researchers hunker down or flee, backlash against Russian science builds By R. Stone

943 Regulator halts assembly of fusion reactor ITER must meet safety concerns before welding giant tokamak sections By D. Clery

944 Mammoth mangrove bacterium has complex cell “Eye-opening” discovery challenges evolutionary thinking on microbes By E. Pennisi

Black physics Ph.D.s are more than twice as likely as other groups to teach in high schools and community colleges. For many, it’s a mission By A. Smart

967 Michigan’s surprising path to diversity Black graduate students find a nurturing culture in its applied physics program By J. Mervis SEE ALSO

Impact of climate change will be worst for the natural world and humanity’s most vulnerable By P. Voosen

EDITORIAL p. 937 PODCAST

949 China quietly plans a pivot from ‘zero COVID’

The Newton’s cradle is often used to demonstrate the conservation of momentum and energy in U.S. college physics courses. But Black students are rare in those classes and, despite decades of efforts to increase Black representation in U.S. physics, the problem is getting worse. A special News section examines why, and ways of reversing the trend. See page 950. Illustration:

Scientists are studying how to live with the virus while avoiding a crisis like in Hong Kong By D. Normile

INSIGHTS PERSPECTIVES

945 New name won’t fix all flaws in China Initiative, critics worry

970 Anthropogenic influences on bee foraging

Justice Department promises tighter focus on preventing espionage and no profiling of Chinese academics By J. Mervis

Efficient foraging is vital to bee fitness but is challenging in the Anthropocene

SCIENCE science.org

964 Called to teach

ON THE COVER

Nik Richard

By D. Goulson and E. Nicholls 4 MARCH 2022 • VOL 375 ISSUE 6584

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CONTENTS

972 Addicted to dreaming

1011 Asteroids

How does dopamine, the brain’s pleasure signal, regulate the dream stage of sleep?

Pebbles and sand on asteroid (162173) Ryugu: In situ observation and particles returned to Earth S. Tachibana et al.

RESEARCH ARTICLE p. 994

1035

974 Forming roots from shoot

1017 Topological physics

Uncovering the genes responsible for different types of roots will transform aspects of plant agriculture By L. Shaar-Moshe and S. M. Brady RESEARCH ARTICLE p. 993

975 Toward a coherent ultracold chemistry Magnetic fields can be used to change chemical reaction rates by a factor of 100 By S. L. Cornish and J. M. Hutson RESEARCH ARTICLE p. 1006

A synthetic monopole source of Kalb-Ramond field in diamond M. Chen et al.

RESEARCH 987 From Science and other journals REVIEW

Long-range vacuum fluctuations break the integer quantum Hall topological protection By A. Rubio

990 Restoration ecology

REPORT p. 1030

REVIEW SUMMARY; FOR FULL TEXT: DOI.ORG/10.1126/SCIENCE.ABE0725

Process conditions allow a titanium catalyst to add just two ethylene molecules to an a-olefin By M. S. Rosen REPORT p. 1021

Soil microbiota as game-changers in restoration of degraded lands O. Coban et al.

Despite recent progress, challenges remain By L. Tang BOOKS ET AL.

982 Life-changing biology Genetically engineered genomes will disrupt the bioeconomy and rewrite human nature— are we ready? By A. Woolfson

983 The lasting legacy of John von Neumann

Elongation and branching of a-olefins by two ethylene molecules T. Dietel et al.

1025 Magnetism Topological magnon band structure of emergent Landau levels in a skyrmion lattice T. Weber et al.

1030 Quantum optics Breakdown of the topological protection by cavity vacuum fields in the integer quantum Hall effect F. Appugliese et al. PERSPECTIVE p. 976

RESEARCH ARTICLES

991 Genetics Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly H. Li et al. RESEARCH ARTICLE SUMMARY; FOR FULL TEXT: DOI.ORG/10.1126/SCIENCE.ABK2432

POLICY FORUM

979 A role for funders in fostering China’s research integrity

1021 Catalysis PERSPECTIVE p. 978

IN BRIEF

976 A new Hall for quantum protection

978 Catalyst coaxed to make branched a-olefins

REPORTS

992 Neurogenomics A single-cell atlas of the normal and malformed human brain vasculature E. A. Winkler et al. RESEARCH ARTICLE SUMMARY; FOR FULL TEXT: DOI.ORG/10.1126/SCIENCE.ABI7377

993 Plant science A conserved superlocus regulates above- and belowground root initiation M. Omary et al. RESEARCH ARTICLE SUMMARY; FOR FULL TEXT: DOI.ORG/10.1126/SCIENCE.ABF4368 PERSPECTIVE p. 974

1035 Molecular knots Vernier template synthesis of molecular knots Z. Ashbridge et al.

1041 Coronavirus Broad anti–SARS-CoV-2 antibody immunity induced by heterologous ChAdOx1/ mRNA-1273 vaccination C. I. Kaku et al.

1048 Coronavirus Structures of the Omicron spike trimer with ACE2 and an anti-Omicron antibody W. Yin et al.

1053 Epigenetics Highly enriched BEND3 prevents the premature activation of bivalent genes during differentiation J. Zhang et al.

994 Neuroscience

DEPARTMENTS

By D. Greenbaum and M. Gerstein

Rapid eye movement sleep is initiated by basolateral amygdala dopamine signaling in mice E. Hasegawa et al.

937 Editorial

LETTERS

PERSPECTIVE p. 972

984 Greek roadless policy: A model for Europe

1000 Synthetic genomics

A new biography seeks to reacquaint readers with the once widely celebrated scientist

By V. Kati et al.

984 Iran’s agricultural waste By R. Akbari and M. Nasrollahzadeh

Transcriptional neighborhoods regulate transcript isoform lengths and expression levels A. N. Brooks et al.

1006 Ultracold chemistry

985 China’s dams threaten the Sichuan taimen

Control of reactive collisions by quantum interference H. Son et al.

By D. Tong

PERSPECTIVE p. 975

Dismantle racism in science By E. O. McGee BLACK PHYSICISTS SECTION p. 950

939 Editorial Biden doesn’t get it By H. H. Thorp

1062 Working Life My writing journey By Y. Tong Science Careers .......................................1059

SCIENCE (ISSN 0036-8075) is published weekly on Friday, except last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue, NW, Washington, DC 20005. Periodicals mail postage (publication No. 484460) paid at Washington, DC, and additional mailing offices. Copyright © 2022 by the American Association for the Advancement of Science. The title SCIENCE is a registered trademark of the AAAS. Domestic individual membership, including subscription (12 months): $165 ($74 allocated to subscription). Domestic institutional subscription (51 issues): $2212; Foreign postage extra: Air assist delivery: $98. First class, airmail, student, and emeritus rates on request. Canadian rates with GST available upon request, GST #125488122. Publications Mail Agreement Number 1069624. Printed in the U.S.A. Change of address: Allow 4 weeks, giving old and new addresses and 8-digit account number. Postmaster: Send change of address to AAAS, P.O. Box 96178, Washington, DC 20090–6178. Single-copy sales: $15 each plus shipping and handling available from backissues.science.org; bulk rate on request. Authorization to reproduce material for internal or personal use under circumstances not falling within the fair use provisions of the Copyright Act can be obtained through the Copyright Clearance Center (CCC), www.copyright.com. The identification code for Science is 0036-8075. Science is indexed in the Reader’s Guide to Periodical Literature and in several specialized indexes.

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science.org SCIENCE

ILLUSTRATION: ZOE ASHBRIDGE/UNIVERSITY OF MANCHESTER

By E. Arrigoni and P. M. Fuller

EDITORIAL

Dismantle racism in science

W

illiam Shockley Jr. of Stanford University was an American physicist who jointly won the 1956 Nobel Prize in Physics for his role in inventing the transistor. Modern computing and communications would have been unthinkable without Shockley’s work. But the Nobel laureate was also a proponent of eugenics and firmly committed to the racial inferiority of Black people. Shockley was not just a physicist who held racist views. He was part of a wider academic system that then, and now, perpetuates racial inequality. The persistence of this biased ecosystem means that dismantling it will require unrelenting tenacity by all of academia. I’ve spent years studying how racism operates in science, technology, engineering, and mathematics (STEM) fields, especially in higher education institutions in the United States. Structural inequities perpetuate tendencies that discriminate against STEM faculty of color and stifle their progress. For example, institutions often expect these faculty to lead the charge when it comes to anti-racist efforts. They are approached to mentor students of color, to serve on diversity committees, and to speak on panels about diversity—activities that are tangential to their research. Although many people of color in STEM have a fervent desire to make their field and workplace more equitable, pressure to perform duties unrelated to their research creates a service burden that many of their white colleagues do not bear. By contrast, white faculty are busy advancing their research, thereby earning grants, joining collaborations, and publishing papers that bolster their careers. This imbalance disadvantages faculty of color in tenure and promotion decisions. People of color who do “make it” in STEM disciplines often do so while taking on a superhuman workload that is unfair and unsustainable. Moreover, to function in the present biased ecosystem, underrepresented people of color are pressured to conform to a predominantly white male culture and are discouraged from bringing their authentic selves into the workplace. For example, STEM faculty of color are encouraged to remove any language about racial justice, including diversity, equity, and inclusion, in their grant proposals. Additionally, some alter their appearance, demeanor, and speech to assimilate into—and survive in—the mainstream STEM culture. This stress is a burden and humiliation that can take a psychological toll.

Because there are fewer people of color in university leadership positions, there are fewer leaders who are cognizant of these impediments. As a result, the barriers for people of color in STEM are rarely addressed because leaders’ priorities are elsewhere. To change the situation, the dominant culture needs to assume the burden of providing remedies. In the field of physics, for example, people of color are called upon to figure out how to “create change,” rather than those who benefit from the status quo. STEM ecosystems need to distribute this responsibility to everyone, especially to those faculty who hold the power and privilege to implement compelling and sustainable changes. Doing this involves the most influential people in efforts to innovate and restructure spaces in ways that support STEM equity. Faculty of color across academia often take on students of color who are outside of their departments but who desperately need mentorship. Institutions need to recognize when faculty of color are functioning as de facto university leaders and reward these staff with the titles, compensation, and resources appropriate to the responsibilities they have assumed. Higher education must also recruit more faculty of color. This is the most powerful way STEM departments can demonstrate that racial equity and diversity are a priority. Students, faculty, and administrators who are women of color experience both raced and gendered forms of abuse in academia. Hostile environments span from negative comments about their abilities, qualifications, and performance to sexual harassment. In my research, I have found that Black women have great difficulties convincing their professors and colleagues that they are worthy of belonging in STEM. Instead of creating programs to “fix” students, how about coaching STEM faculty and administrators on the toxicity associated with racially unwelcoming conditions and the particular effects it has on women of color? Having more women of color leadership in STEM academia would be a strong start. Anti-racism work is hard work, but unless actions move beyond simply tossing about diversity and equity buzzwords, people of color will remain underrepresented in STEM fields. And the United States will continue to miss out on the STEM talent and innovation that exist within communities of color. –Ebony Omotola McGee

PHOTO: S. KRUPNICK

“…the barriers for people of color in STEM are rarely addressed…”

Ebony Omotola McGee is a professor of Education, Diversity, and STEM Education in the Department of Teaching and Learning at Peabody College, Vanderbilt University, Nashville, TN, USA. ebony.mcgee@ vanderbilt.edu

Published online 1 March 2022; 10.1126/science.abo7849

SCIENCE science.org

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937

Rutgers University: R&D with Impact Committed to research and development driven by a relentless pursuit of excellence

$907.9 million Research grants and sponsored programs

1187

Active technologies to license and market

$720 million

Research and development expenditures

1000+

Companies partner with Rutgers researchers

Top 5 Direct Federal Sponsors of Rutgers Research

Rutgers exceeds all NJ colleges and universities combined in R&D expenditures

■ National Institutes of Health $229.7M ■ National Science Foundation $56.9M ■ U.S. Health Resources and Services Administration $17.2M ■ U.S. Department of Education $9M ■ National Oceanic and Atmospheric Administration $6.8M

Recent Outcomes with Global Impact ■ Invented COVID-19 diagnostic tests and serving as COVID-19 vaccines clinical trials site ■ Pioneering use of underwater gliders for hurricane intensity forecasting

Top 100

■ Lead authorship of Intergovernmental Panel on Climate Change Reports

World’s Most Innovative Universities —Reuters

■ Invented clean manufacturing technology for polymers and other monomers ■ Co-lead investigator of large-scale urban test site for next-gen Wi-Fi

excellence.rutgers.edu Annual data current as of January 2022

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EDITORIAL

Biden doesn’t get it

I

t has been a rough 2 weeks for the US science community. After 4 years of bludgeoning by the Trump administration, hope resurged a year ago as a new White House promised to value science. But there have been missteps, the most recent taking place on the heels of another blunder that many saw coming. Eric Lander, who just stepped down as President Biden’s science adviser and director of the Office of Science and Technology Policy (OSTP), was a prominent research figure with a well-known record of bullying and callous actions. With the notable exception of the 500 Women Scientists organization, the scientific community was embarrassingly silent about Lander’s nomination. Not surprisingly, he is out of the White House because of the same behavioral issues. And yet, in another tone deaf move, the administration just named Francis Collins, the recently retired director of the National Institutes of Health (NIH), as Lander’s interim replacement as science adviser while asking Alondra Nelson, the OSTP’s deputy director for science and society and an experienced administrator and scholar, to temporarily direct OSTP. Apparently, Biden doesn’t think Nelson is capable of doing both jobs. I disagree and am not staying silent this time. The decision to choose Collins for the presidential adviser aspect of the job may appear a strategic move to advance Biden’s biomedical agenda, but the decision to keep Nelson out of this role reflects a chronic ill in America—inequity at the highest levels of leadership. Lander’s vacated position and Nelson’s presence at OSTP already presented a clear opportunity to put someone in the combined role who represents the future of American science. Certainly, Francis Collins has been a major science policy figure in the US for many years. Through his dogged determination and political skill, he pushed the human genome project to completion and took the helm of the NIH, serving three presidents. Through his masterful abilities to engage with Congress, he reliably secured increases in NIH funding year after year. But the end of his tenure brought problems that weren’t adequately resolved. He was unable to dismantle an outdated grant assessment process that reinforces a low funding rate for Black applicants. And NIH caved to political pressure around the China

Initiative, which sought to satisfy the Trump administration’s anti-Asian tendencies by sending letters to universities to disrupt legitimate scientific collaborations with China. At some point, the scientific community must stand up against anti-Asian racism and injustice in the funding of Black scientists, among other discrimination in the scientific enterprise. That time has to be now, and what is needed is a new cadre of scientific leaders in Washington, DC, who can build a scientific agenda where inclusion of individuals who reflect the diverse makeup, talents, and interests of society is prioritized. Appointing someone as the nation’s science adviser— even on a temporary basis—who has already enjoyed the privilege of leading for many years sends a message that the White House doesn’t care, especially when so many new and diverse leaders are available. It’s hard not to conclude that the administration has gotten what it needs out of science and has lost interest. Given the sluggish pace with which science appointments have been made by the administration, this latest arrangement may last for some time. Alondra Nelson is one of the visionary leaders in American science today. She was the first Black professor to receive tenure in sociology at Columbia University, has held numerous challenging administrative roles, and currently occupies the Harold F. Linder Chair in the School of Social Science at the Institute for Advanced Study. When invited to join OSTP, Nelson stated that “as a Black woman researcher, I’m keenly aware of who has been missing from the room.” After 2 years of witnessing the scientific community succeed at developing vaccines to combat the pandemic but fail at getting full acceptance by the community, it’s clear that Nelson has the expertise that is gravely needed for all of science. It’s time for the scientific community to commit to a vision of the future where young scientists can get a fair share of funding, where equity for women and people of color is prioritized over protecting processes that reinforce injustices of the scientific enterprise, and where leaders in Washington, DC, represent the future, not the past. –H. Holden Thorp

H. Holden Thorp Editor-in-Chief, Science journals. [email protected]; @hholdenthorp

PHOTO: CAMERON DAVIDSON

“Lander’s vacated position and Nelson’s presence at OSTP already presented a clear opportunity…”

Published online 18 February 2022; 10.1126/science.abo6922

SCIENCE science.org

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939

NEWS

“

Fifty percent is a failing grade. It’s an F. Someone with the resources of Facebook should be aiming for an A.

”

Imran Ahmed of the Center for Countering Digital Hate, on its finding that Facebook is failing to label about half the posts that promote articles from publishers of climate misinformation.

IN BRIEF Edited by Jeffrey Brainard

Many schools that have mandated wearing masks are considering making them optional, following new U.S. government guidance.

COVID-19

T

he often-acrimonious U.S. debate over wearing masks entered a new phase last week when the Centers for Disease Control and Prevention (CDC) loosened its guidance and suggested about 70% of the population could jettison them. The turnabout comes as the Omicron wave ebbs and scientists consider how to recalibrate public health precautions, given the mostly mild or moderate disease the virus variant causes in vaccinated people. CDC’s new approach relies on regional COVID-19 hospital admission

Nobelists denied CRISPR patent | The team that won a Nobel Prize for its groundbreaking creation of the CRISPR genome editor lost a key patent battle this week. An appeals board of the U.S. Patent and Trademark B I O T E C H N O L O GY

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rates, the percentage of hospital beds filled by those patients, and new cases per 100,000 people during the past week. Previously, case numbers had played the starring role. Some scientists praised the agency’s change; others criticized it because the U.S. population includes many vulnerable people, like those with compromised immune systems, and children under age 5, for whom COVID-19 vaccines aren’t yet available. State and local health authorities in the United States determine mask wearing rules but often turn to CDC for guidance.

Office ruled that a rival group led by the Broad Institute of MIT and Harvard convincingly showed it first “reduced to practice” the use of CRISPR as an editor of eukaryotic cells in October 2012. Nobel laureates Jennifer Doudna and Emmanuelle Charpentier and their colleagues first

described using CRISPR to edit specific DNA in 2012, but the group, known as the CVC, struggled to make it work in eukaryotic cells. The patents for CRISPR in eukaryotic cells represent a huge financial windfall because these are the foundation for human medicines. The CVC, which science.org SCIENCE

PHOTO: SCOTT MCINTYRE/THE NEW YORK TIMES

CDC relaxes guidance on wearing masks in public

lost an earlier patent battle over a similar claim, retains 40 other U.S. patents related to CRISPR and an intellectual property portfolio in 30 countries. A CVC representative said the team “is considering various options to challenge this decision.”

Panel to mull risky virus research | In the wake of the coronavirus pandemic, the U.S. government is revisiting its oversight of “gain-of-function” studies that modify pathogens in ways that could make them more harmful to people. This week, the White House and National Institutes of Health asked the National Science Advisory Board for Biosecurity to review the scope and effectiveness of policies for research on “enhanced potential pandemic pathogens.” The assessment could examine whether controversial coronavirus experiments previously funded by the United States in China fall under that definition and should have received stricter scrutiny. The board will also look at existing policies on “dual-use research of concern” involving pathogens that could be misused to cause harm. Some board members cautioned against overly restrictive rules that could shut down research essential to fighting pandemics. But some critics have called existing policies too permissive. A report is due by December. BIOSECURITY

Intellia Therapeutics and Regeneron Pharmaceuticals announced that 1 month after receiving an injection of fat particles carrying messenger RNA for the CRISPR enzyme and a guide RNA sequence, six people with transthyretin amyloidosis, an inherited, progressive disease, had a drop in blood levels of a misfolded liver protein that can damage the heart and nerves. The companies said in a 28 February press release and webcast with investors that the reductions in levels—ranging from 41% to 93%, depending on the CRISPR dose—have remained stable in the six original participants and nine more, all treated between 2 and 12 months ago. The researchers do not know yet whether the patients’ neuropathy symptoms have improved.

Colonialism in fossil studies | Two basins—one in northeastern Brazil, the other in northern Mexico—have provided dozens of well-preserved fossils from the Jurassic and Cretaceous periods for research. But many journal articles about those finds, published during the past 3 decades, serve as glaring examples of what a new analysis calls “paleontological colonialism.” Examining about 200 such published studies, a team of scientists including ones from those countries found that approximately half the studies were led by foreign RESEARCH ETHICS

Subvariant proves no more severe | BA.2, a more contagious version of the already highly infectious Omicron SARS-CoV-2 variant, does not make people sicker than the original Omicron, the World Health Organization (WHO) concluded last week, based on data from three countries where the subvariant has become dominant. Both a 19 February preprint from South Africa’s National Institute for Communicable Diseases and a 25 February report by the UK Health Security Agency showed no increase in the risk of hospitalization for people who carried BA.2, compared with those infected by the original Omicron variant, known as BA.1. And in a 22 February preprint, the Danish public health agency reported that reinfection by BA.2 did not lead to worse symptoms than an initial BA.1 infection, which were mild or moderate. C OV I D -1 9

PHOTO: RENAN BANTIM

| Children ages 5 to 11 inoculated with the Pfizer COVID-19 vaccine lose most protection against infection by SARS-CoV-2 in as little as 1 month, although defense against hospitalization seems to persist, new evidence shows. The news disappointed public health specialists because the Pfizer vaccine is the only one authorized in the United States for that age group. The findings drew on New York state medical databases and compared more than 1 million vaccinated 5- to 17-year-olds with their unvaccinated peers. Among 5- to 11-year-olds who received two doses of Pfizer’s messenger RNA–based vaccine by the second half of December 2021, the vaccine’s effectiveness against infection dropped from 65% to 12% a month later; in teenagers vaccinated in that same period, the decline was smaller, from 76% to 56%, according to the analysis by researchers at the New York State Department of Health. The authors of the study, posted on 28 February as a preprint on medRxiv, suggest younger children may have lost more protection because they received smaller doses of the Pfizer vaccine than teenagers. C OV I D -1 9

| LGBTQ scientists who don’t disclose their sexual orientation in the workplace publish fewer papers than their peers, a study has found. Researchers surveyed 1745 academic scientists in 2016, 1093 of whom identified as a sexual minority, asking them questions about their publishing history and openness in the workplace. Based on the information each provided, straight men and openly gay or bisexual men published the most papers, whereas LGBTQ individuals whose colleagues weren’t aware of their sexual orientation published the fewest. The authors of the study, which appeared this week in PLOS ONE, say the publishing gap may reflect the challenges that LGBTQ individuals face when working in environments in which they’re not comfortable sharing their identity. DIVERSITY

THERAPEUTICS

SCIENCE science.org

Vaccine’s protection for kids dives

LGBTQ disclosure linked to papers

Edited gene’s effect persists | The first team to edit a disease gene by deploying CRISPR, the DNA scissors, within the body, reported this week that levels of a toxic protein stayed low for as long as 1 year, suggesting long-term benefits. In June 2021,

researchers. Of those, half lacked any local researchers as co-authors or showed no evidence of any collaboration with them— even though Brazil and Mexico require such partnerships. Many publications did not state whether the authors obtained permits for fieldwork, and many of the specimens described in the papers had been illegally deposited in foreign collections, according to the study, published on 2 March in Royal Society Open Science.

Brazilian police seized fossils illegally offered for sale and presented them to a museum at a 2021 event.

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IN DEP TH

SCIENTIFIC COMMUNITY

War in Ukraine poses stark choices for scientists By Richard Stone

O

n 24 February, as Russian troops poured across the border in an unprovoked invasion of Ukraine, Sergei Mosyakin, director of the Institute of Botany in Kyiv, set out with a few key staff to secure the institute and its National Herbarium, which holds more than 2 million specimens representing the wealth of Ukraine’s floral and fungal diversity. Several kilometers to the south, Fedor Danevich and six colleagues at the Institute for Nuclear Research had joined an online workshop for a multinational physics project in South Korea. The Zoom call included Russian physicists. “One said that he is sorry for the war,” Danevich recalls. The other Russians kept silent. In the weeks leading up to the invasion, many Ukrainian scientists had dismissed Russia’s military buildup on their border as bluster. Now, their lives have been turned upside down as they make fateful decisions about whether to dig in or flee to other European nations that have stepped up to offer accommodations and job prospects. As Russian forces bombarded and sought to encircle Kyiv, Mosyakin and some fellow 942

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botanists hunkered down at home, while others took refuge with relatives in the countryside. Danevich says he intended to stay in Kyiv, but his son persuaded him to gather vulnerable family members and seek shelter in Budapest, Hungary. As Science went to press, they had made it as far as Romania. Condemnation of the invasion has rained down on Russia from many quarters, and a rising chorus is calling on the West to sever ties with Russian scientists. “I’m sitting now with my 86-year-old mother, who is a prominent biochemist, listening to the sounds of battle some 20 kilometers to the west, and waiting for the next bombardment,” says Maksym Strikha, a physicist and former top official in Ukraine’s science ministry. “Could you imagine asking a Polish physicist, surrounded and bombed in Warsaw in September 1939, whether it would be fair to maintain scientific diplomacy with scientists in Nazi Germany?” The repercussions in international science are already being felt. The European Space Agency (ESA) announced on 28 February that international sanctions against Russia and “the wider context” are likely to delay by at least 2 years the launch of a Mars rover, part of the ExoMars astrobiology mission

jointly sponsored by Russia and ESA. And the Massachusetts Institute of Technology (MIT) is pulling out of a key collaboration with a Russian university, the Skolkovo Institute of Science and Technology (Skoltech), which MIT had helped found. Last week, as Russian troops crossed the border, Ukrainian scientists pleaded for more drastic steps. In an open letter posted on 27 February, the Council of Young Scientists at the Ministry of Education and Science of Ukraine called on the European Commission to “urgently suspend all kinds of international collaboration with Russian institutions,” including ending Russian scientists’ participation in Horizon Europe, the flagship research fund. They also urged that Russia be expelled from two premier international R&D ventures—the experimental ITER fusion reactor in France and the CERN particle physics research center in Switzerland. For now, Russia remains part of Horizon Europe and other European funding schemes. And the nine-nation collaboration Danevich is involved in to examine the nature of the neutrino—the Advanced Mobased Rare process Experiment (AMoRE) at South Korea’s Yangyang Underground science.org SCIENCE

PHOTO: GLEB GARANICH/REUTERS

As Ukrainian researchers hunker down or flee, backlash against Russian science builds

NE WS

Smoke and flames rose over Kyiv, Ukraine, during last week’s Russian invasion.

Laboratory—is proceeding. In an email to collaborators, AMoRE’s spokespeople described its leaders’ “determination to carry out our program during this difficult period.” But the collateral damage to Russia’s scientific enterprise is mounting. In addition to the effects on ExoMars and Skoltech, the United States imposed fresh sanctions on technology transfers to Russia, which the Russian space agency claimed could threaten operation of the International Space Station. After Germany’s research ministry ordered a suspension of collaboration with Russia, the Max Planck Institute for Extraterrestrial Physics turned off the main instrument on Russia’s Spektr-RG research satellite: the German-run eRosita x-ray survey telescope, meant to probe the universe’s large-scale structure. And math societies in several countries announced last week they will not participate in the International Congress of Mathematicians, which was slated for St. Petersburg, Russia, in July but now plans to go virtual. Within Russia itself, some scientists sought to show solidarity with Ukraine. More than 5400 Russian scientists and science journalists signed a letter calling the war “a step to nowhere” that will turn Russia into a pariah. The invasion, the 24 February letter says, “means that we scientists will no longer be able to do our job normally: after all, conducting scientific research is unthinkable without cooperation with colleagues from other countries.” Posted to the independent science news site TrV-Nauka, the letter is in SCIENCE science.org

part an appeal to the international community “that any actions to punish Russia are weighted in such a way as to not punish the very people who object to what Russia is doing,” says Mikhail Gelfand, a bioinformatics specialist who helps edit TrV-Nauka. (He notes that he is not speaking on behalf of his employer, Skoltech.) And climatologist Oleg Anisimov, head of Russia’s delegation to the United Nations’s Intergovernmental Panel on Climate Change, apologized for the invasion during a 27 February meeting, according to news reports. Russian scientists may face peril for speaking out on Ukraine’s behalf. The office of Russia’s prosecutor general has declared that anyone caught providing assistance to a nation or organization acting against the Russian Federation could be charged with treason. Authorities have arrested hundreds of street protesters, including Skoltech professor Georgii Bazykin, a prominent evolutionary geneticist who was detained on 27 February after holding up a handwritten sign in downtown Moscow saying, “Stop the War Today.” He was released pending trial. Bazykin later tweeted that his plight is a “minor inconvenience compared to what people of Ukraine face now, or what the world including Russia will face if [President Vladimir] Putin has his way.” In Europe, a number of institutions have offered refuge to Ukrainian scientists, some of whom have joined an exodus of more than 500,000 people from the besieged country. The Polish Young Academy, part of the Polish Academy of Sciences (PAN), has lined up scores of institutes and universities willing to host refugee scientists. “We want to find them jobs in their expertise,” says Jacek Kolanowski of PAN’s Institute of Bioorganic chemistry. So far, they’ve secured positions for a psychologist in Warsaw, a cancer researcher in Lublin, and a law professor in Poznan´. In Germany, the Alexander von Humboldt Foundation’s Philipp Schwartz-Initiative, which helps find succor for scholars at risk, has invited Ukrainian scientists to apply for funding with German partners. “Our lab in Berlin will support researchers who had to flee Ukraine,” tweeted Matthias Rillig, an ecologist at the Free University of Berlin. Many Ukrainian scientists have vowed to stay and defend their homeland. “Virtually the entire population has taken up arms,” says Oleg Krishtal, a neurophysiologist with the Bogomoletz Institute of Physiology, who is sticking it out in Kyiv. Mosyakin says that although he is a mixture of at least four nationalities, “I am a Ukrainian, period.” And most Ukrainians he knows “are not afraid to die for their native land.” j

FUSION

Regulator halts assembly of fusion reactor ITER must meet safety concerns before welding giant tokamak sections By Daniel Clery

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rance’s nuclear regulator has ordered ITER, an international fusion energy project, to hold off assembling its doughnut-shaped reactor, called a tokamak. On 25 January, France’s Nuclear Safety Authority (ASN) sent a letter halting work until ITER can respond to safety concerns. ITER staff say they intend to satisfy ASN so they can begin welding by July—and still open in 2025. Fusing hydrogen isotopes under extreme temperatures generates energy. But no fusion reactor has produced more heat than it consumes. The $25 billion ITER project is designed to demonstrate net energy output, but not until 2035. One ASN concern is over the 1.5-meterthick slab of concrete on which the reactor sits. ASN wants reassurance that the loads on the slab are within safety limits. A second concern is protecting staff and the public from radiation. Thick concrete walls in the reactor building should stop high-energy neutrons produced during operation. But existing “radiological maps do not make it possible to demonstrate control of limiting exposure,” ASN writes. ITER Director-General Bernard Bigot says most nuclear facilities only produce a 2D model of potential exposures. ASN wants more evidence that ITER’s 3D model is as robust as the simpler one. A third concern is over slight deformities discovered in two of the nine 11-meter-tall sections that will make up the tokamak. ITER staff developed a fix that would involve robotic and human welders, but ASN is not convinced. Bigot hopes ASN will be satisfied by tests of the welding system on a full-scale mockup. Bigot says he understands ASN’s desire to be careful with a machine that will be the first of its kind. “They want to understand very precisely the safety risks,” Bigot says. “So it’s not surprising they’re taking a little more time.” j 4 MARCH 2022 • VOL 375 ISSUE 6584

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BIOLOGY

Mammoth mangrove bacterium has complex cell “Eye-opening” discovery challenges evolutionary thinking on microbes giant sulfur-eating microbe roughly the size of a poppy seed off Namibia’s coast. Its y definition, microbes are so small cellular contents are squished up against they can only be seen with a microits outer cell wall by a giant water- and scope. But a newly described bactenitrate-filled sac. The sac means the bacrium living in Caribbean mangroves teria’s essential molecules only need to difshatters that rule. Its threadlike single fuse short distances because “only [along cell can be the length of a peanut—up the edge] is the cell living,” says Carvalho, to 2 centimeters long, 5000 times bigger who worked on this group of bacteria. than many other microbes. And whereas The new mangrove bacterium also has a the genetic material of other bacteria floats huge sac—presumably of water—that takes freely within the cell, the huge genome of up 73% of its total volume. That similarthis giant is encased in a membrane, like ity and a genetic analysis led the research that of more sophisticated cells including team to place it in the same genus as most those in the human body. of the other microbial giants and propose Unveiled in a preprint posted bioRxiv calling it Thiomargarita magnifica. last month, the organism drew aston“What an excellent name!” says Andrew ished reactions. “FantasSteen, a bioinformatician at tic and eye-opening,” says the University of Tennessee, Victor Nizet, a physician sciKnoxville, who studies how entist at the University of microorganisms affect geoCalifornia, San Diego, who chemical cycles. “Reading studies infectious diseases. about it makes me feel ex“When it comes to bacteria, I actly the same way as when never say never, but this one I hear about an enormous for sure is pushing what we dinosaur.” thought was the upper limit Its DNA proved extraordi[of size] by 10-fold,” adds nary as well. When researchVerena Carvalho, a microers at the Department of biologist at the University of Energy Joint Genome InstiMassachusetts, Amherst. tute sequenced it, they found Aside from breaking size rethe genome was enormous, cords, the bacterium, with its with 11 million bases harborelaborate internal structure, ing some 11,000 clearly dis“could be a missing link in the tinguishable genes. Typically, evolution of complex cells,” bacterial genomes average says Kazuhiro Takemoto, a about 4 million bases and computational biologist at the A new bacterium’s single-cell filaments are visible next to a dime. about 3900 genes. Kyushu Institute of TechnoBy labeling the DNA with logy. Researchers have long thought that cludes two membrane sacs, one of which fluorescent tags, Volland determined the only eukaryotes, from yeast to most forms contains all the cell’s DNA, like the nucleus bacterium’s genome was so big because it of multicellular life, package their DNA in a of a eukaryotic cell. includes more than 500,000 copies of the nucleus and compartmentalize various cell “Perhaps it’s time to rethink our definisame stretches of DNA. No one really knows functions into vesicles called organelles. tion of eukaryote and prokaryote!” says why. Protein production factories called riWhen Olivier Gros, a marine biologist Petra Levin, a microbiologist at Washingbosomes nestled inside the DNA-filled sac at the University of the French Antilles, ton University in St. Louis. as well, likely making the translation of a Pointe-à-Pitre, came across the strange orThe other sac may be the key to the microgene’s code into a protein more efficient. ganism growing as thin filaments on debe’s size. Microbiologists used to think It’s yet another way T. magnifica defies caying mangrove leaves in a local swamp a bacteria had to be small, in part because preconceptions about bacteria, says Chris decade ago, he had no idea what it was. Not they rely on diffusion, which only works Greening, a microbiologist at Monash until 5 years later did he and colleagues exover short distances, to carry out respiraUniversity, Clayton. amine its DNA and realize the filaments tion, transport nutrients into and around “All too often, bacteria are thought of as were actually bacteria. And they didn’t the cell, and get rid of toxins. Eukaryotes, small, simple, ‘unevolved’ life forms—soappreciate how special the microbes were in contrast, actively transport molecules called ‘bags of proteins,’” Greening adds. until more recently, when Gros’s graduthrough molecular pumps and channels. “But this bacterium shows this couldn’t be ate student Jean-Marie Volland took up Then in 1999, researchers discovered a much further from the truth.” j

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the challenge of trying to characterize them. Other microbes, such as slime molds and blue-green algae, form similar filaments, made up of many cells stacked together. But a variety of microscopy and staining methods made it clear the mangrove filaments were each just one enormous cell. This “was something we didn’t believe … at first,” recalls Volland, now a marine biologist at Lawrence Berkeley National Laboratory. The largest specimen of this new bacterium so far stretched 2 centimeters, but Carvalho thinks they could grow even bigger if not trampled, eaten, blown by wind, or washed away by a wave. Volland and his colleagues also found that each cell in-

science.org SCIENCE

PHOTO: TOMÁŠ TYML

By Elizabeth Pennisi

Assistant Attorney General Matthew Olsen has renamed a controversial Department of Justice initiative that has prosecuted academic scientists.

SCIENCE AND SECURITY

New name won’t fix all flaws in China Initiative, critics worry Justice Department promises tighter focus on preventing espionage and no profiling of Chinese academics By Jeffrey Mervis

PHOTO: WIN MCNAMEE/GETTY IMAGES

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niversity scientists and civil rights groups in the United States are offering qualified praise for the federal government’s decision last week to rename and revise the China Initiative, a controversial, 3-year-old law enforcement campaign intended to prevent the Chinese government from stealing U.S.funded technologies. “Dropping the name is good,” says Steven Pei, an electrical engineer at the University of Houston who has been a prominent advocate for reforming an initiative critics say has unfairly targeted U.S.-based scientists of Chinese origin and improperly subjected researchers who made paperwork errors to criminal prosecution. “But the real issue is how the new policy will be implemented.” Pei and other observers also worry the China Initiative has already done lasting damage to international research collaborations. The same suspicions that fed the initiative, they say, are driving Congress toward adopting tougher disclosure rules for U.S. researchers working with foreign partners and banning certain collaborations altogether. SCIENCE science.org

Since its launch in 2018 under thenPresident Donald Trump, the China Initiative has resulted in criminal charges against some two dozen academic scientists, typically for failing to tell U.S. funding agencies about their financial ties to Chinese institutions. Most of the defendants were of Chinese origin. Although several pled guilty and were sentenced to prison terms, prosecutors dropped other cases and only one scientist, Harvard University chemist Charles Lieber, has been convicted by a jury. Both that checkered record and sharp criticism from many quarters led to a review of the initiative by President Joe Biden’s appointees at the Department of Justice (DOJ). Last week, Assistant Attorney General Matthew Olsen, who took over DOJ’s national security division in November 2021, announced the initiative will now be called “a strategy for countering nation-state threats.” The new name, Olsen explained during a 23 February speech at George Mason University, recognizes that the biggest danger to U.S. national security comes from foreign governments, not individuals or a particular ethnic group. China stands at the top of that list, Olsen said, pointing to its ongoing cyber-

attacks and silencing of dissent as well as several explicit acts of economic espionage. He cited a recent speech by FBI Director Christopher Wray that described China’s behavior as “more brazen and threatening than ever before.” In another change, Olsen said federal prosecutors will look more carefully at whether academic scientists accused of disclosure violations should face criminal charges. Science advocates have argued that most such lapses should be treated as a form of scientific misconduct and addressed with civil or administrative penalties. Olsen appeared to tacitly agree, referring to statements from academic leaders that DOJ’s “pursuit of certain research grant fraud cases … can lead to a chilling atmosphere … that damages the country’s scientific enterprise.” Olsen also acknowledged criticism from civil rights advocates that the Trump-era label had “fueled a narrative of intolerance and bias.” But he did not apologize for any missteps that might have occurred, a silence that rankles many scientists. “If you don’t admit that you’ve done something wrong, then how can you prevent it from happening again?” asks Pei, a co-organizer of the nonprofit Asian Pacific American Justice Task Force, which has highlighted the plight of scientists it argues were unjustly prosecuted. Pei and others would like DOJ to conduct a blanket review of all pending cases. “That would go a long way toward winning back the trust of the Asian and scientific communities,” Pei says. In his speech, Olsen said the new strategy would be applied “going forward” and not used to review existing cases. Even so, the government has agreed to a 4-month delay in the case of physicist Zhengdong Cheng, a tenured professor who was fired by Texas A&M University, College Station, after the government accused him of defrauding NASA by not disclosing ties to China in a 2013 grant application. The trial of Cheng, who was detained for 1 year after his August 2020 arrest, was supposed to start on 4 April. But 2 days after Olsen’s speech, Cheng’s lawyer filed an unopposed motion for delay. It states the government and Cheng “have now entered into good-faith negotiations to resolve the prosecution … so that justice may be done.” Olsen also promised to exercise greater oversight over FBI-conducted investigations. Michael German, a former FBI special agent now at New York University’s Brennan Center for Justice, says the China Initia4 MARCH 2022 • VOL 375 ISSUE 6584

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tive emboldened FBI agents to target anyone with ties to China rather than following the traditional practice of gathering evidence of wrongdoing before pursuing a full-blown investigation. “If your boss calls it the China Initiative, then you focus on anyone with connections to China,” German says. That strategy backfired in the prosecution of Anming Hu, a mechanical engineering professor at the University of Tennessee, Knoxville, who was acquitted in September 2021 in a trial in which FBI agents admitted they had found no evidence of economic espionage. German hopes FBI will now put a priority on cases where it suspects espionage or the theft of intellectual property. Pei suggests using a simple metric for determining whether DOJ is following through on its reforms: “Fewer knocks on the doors of academic scientists by FBI agents.” But Margaret Lewis, a China scholar and law professor at Seton Hall University, worries the new policy may do little to protect valuable collaborations with China given the abundant “anti-China rhetoric in Washington.” She notes that both the Senate and the House of Representatives have voted to prohibit any scientist supported by China’s foreign talents programs from receiving federal research dollars—a consensus that makes the ban likely to be included in a massive pending bill aimed at strengthening the ability of the United States to compete with China. If that happens, Lewis fears universities could create administrative “buffer zones” that discourage other kinds of interactions with China, in order to make sure that faculty don’t violate the ban. That prospect troubles Peter Michelson, a physicist at Stanford University. Last year, he helped organize a faculty petition asking Attorney General Merrick Garland to end the China Initiative that has become a template for similar letters from many U.S. campuses. “I’m afraid that university administrators have become increasingly gutless in standing up to government research agencies because they are so dependent on federal funding,” he says. Chinese students—who represent a significant fraction of U.S. graduate students in many technical areas—are increasingly wary of coming to a country they believe doesn’t want them, Michelson adds. “This year [at Stanford] there was a significant drop in the number and quality of Chinese applicants to some of our graduate programs,” he says. Such thorny issues are a big reason many scholars see DOJ’s announcement as only a first step. The name change “recognizes that our concerns were legitimate,” says John Yang, president of Advancing JusticeAAJC, an advocacy group. “But there is a lot more work to be done.” j 946

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COVID-19

Studies bolster pandemic origin in Wuhan animal market Close scrutiny of earliest cases and samples from market suggests virus crossed over from animals sold there By Jon Cohen

Skeptics of the natural-origin theory maintain the market cluster could merely hree new studies offer one indisputbe a superspreader event touched off when able conclusion about the origin of a person infected with a lab-escaped coronaSARS-CoV-2: Despite the passage of virus visited it. But Worobey thinks further 2 years and the Chinese government’s data could make that contention even less lack of transparency, data that can tenable. A more transparent analysis of the shed light on the pandemic’s greatest market’s genetic sampling data, in particumystery still exist. And although these new lar, might identify exactly which species of analyses don’t all reach the same concluanimals sold there carried the virus. sion for how COVID-19 was sparked, each In one study, Worobey and colleagues undercuts the theory that the virus somedescribe two subtly different lineages of how escaped from the Wuhan Institute of SARS-CoV-2 that were found in people Virology, long a focus of suspicions. and environmental samples at the HuaThe studies examine different aspects nan Seafood Market in late 2019, which of the viral spread at the Huanan Seafood they take as a sign that the virus jumped Market in Wuhan, China, the twice from animals to humans city where the first cases were there. Their other study ofdetected. Two international fers a geospatial analysis of efforts build the case that the earliest human cases that SARS-CoV-2 jumped to peopinpoints the market as the ple from infected animals—a “epicenter” of SARS-CoV-2’s zoonotic leap—at the market, emergence, showing both linlikely twice, at the end of 2019. eages infected people who had A third, largely Chinese eflinks to the market or lived fort details early signs of the near it. It also connects the coronavirus in environmenspecific stalls at the market tal samples from the market. where live animals were sold Flo Débarre, CNRS But it suggests the virus was to SARS-CoV-2-infected enviimported there, perhaps from ronmental samples. “Together, outside the country—a conclusion the Unithese analyses provide dispositive evidence versity of Arizona’s Michael Worobey, an for the emergence of SARS-CoV-2 via the evolutionary biologist who is a correspondlive wildlife trade and identify the Huanan ing author of the two international studies, market as the un-ambiguous epicenter of calls “a huge disconnect.” the COVID-19 pandemic,” they conclude. The studies were posted as preprints Worobey and colleagues had hoped to and are not peer reviewed, but scientists, release their preprints in the next week biosecurity experts, journalists, and othbut sped up their plans, choosing a preers are already intensely examining their print server, Zenodo, that posts without details. “I have been brought closer to the any delays, when the Chinese study was zoonosis side with these preprints,” says posted on 25 February on the Research Flo Débarre, an evolutionary biologist Square site. Led by George Gao of the at the French national research agency, Chinese Academy of Sciences and coCNRS, who has followed the origin debate authored by 37 other scientists (one is closely and not thrown her lot with either from Canada), that research—which builds the natural-origin or the lab-leak camp. on data earlier leaked to the media but Evolutionary biologist William Hanage of never officially published—offers the most Harvard University agrees these studies detailed description yet of the environ“will be taken as a blow” to the lab-leak mental samples the Chinese Center for hypothesis. “They substantially move the Disease Control and Prevention obtained needle on the origins in the direction of at the Huanan Seafood Market between the market,” Hanage says. 1 January and 2 March 2020.

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“I have been brought closer to the [natural-origin] side with these preprints.”

science.org SCIENCE

PHOTO: AP PHOTO/DAKE KANG

New analyses build the case that COVID-19 originated at the Huanan Seafood Market in Wuhan, China.

In the new preprint, Gao and colleagues analyzed 1380 samples from 188 animals in the market and the environment, including sewer wells, the ground, feather removing machines, and “containers.” They found SARS-CoV-2 in 73 samples. But because all were from the environment, not the animals themselves, they assert that humans introduced the virus to the market. The authors call the market an “amplifier,” not the source, of SARS-CoV-2. Hewing closely to government assertions on COVID-19’s origin, the preprint by Gao and colleagues notes studies that have reported evidence of SARS-CoV-2 in other countries before it surfaced in Wuhan, making no mention of critiques that attribute that evidence to contamination. It also floats a widely disputed theory that frozen food imported to China might have been the original source. (Authors of the paper, including Gao, did not respond to requests to discuss the work.) The coronavirus lineage analysis from Worobey and colleagues refines an argument posited by Tulane University virologist Robert Garry last year. In data on the early human cases, Garry had identified two forms of SARS-CoV-2, differing by just two mutations, which he argued surfaced at different Wuhan markets in December SCIENCE science.org

2019. The new work, which includes Garry as a co-author and cites evidence from the Gao study, reshapes that scenario significantly. It concludes that both lineages, dubbed A and B, originated at the Huanan Seafood Market and soon spread in nearby neighborhoods. B likely jumped from animals to humans in late November 2019, leading to the first detected case on 10 December, and lineage A a few weeks later, the group concludes. Either way, the team argues the almost simultaneous emergence of two lineages challenges the lab-origin thesis, as it would require two different viruses leaking at roughly the same time. (Gao and colleagues also found both SARSCoV-2 lineages in environmental samples.) The second preprint from the international team builds on a June 2021 Chineseled study that spent 2 years documenting a tick fever disease in mammals for sale at specific stalls in the market. The new study pinpoints for the first time where species susceptible to SARS-CoV-2—including raccoon dogs, hedgehogs, badgers, red foxes, and bamboo rats—were sold and maps those sites to the positive environmental samples, including one from a “container” the authors believe was a cage. “To anyone who really grasps what is in all of those three papers, I think it’s very hard to dis-

miss that this is a very, very, very strong case that this pandemic started at that market,” Worobey says. Others say the evidence is not definitive. “They are interesting studies, but I don’t think they close the case on what happened with the origins of the virus,” says Jesse Bloom, an evolutionary biologist at the Fred Hutchinson Cancer Research Center who has criticized colleagues for too blithely dismissing the lab-origin hypothesis. “I’m especially skeptical of the conclusion that there must have been two zoonotic jumps.” He notes that in about 10% of human transmissions of SARS-CoV-2, the virus acquires two mutations, which means a second lineage could have emerged after the infection of the first human rather than after a second zoonotic jump. Worobey, Garry, and colleagues did a computer simulation that challenges Bloom’s assertion. They modeled many SARS-CoV-2 outbreaks, accounting for how the virus has been shown to mutate, and assessed each simulation’s results against the actual viruses sequenced from Wuhan COVID-19 cases through 23 January 2020. They found there was only a 3.6% chance that a single lineage of SARS-CoV-2 starting in one person could have produced the second lineage and the later known sequences. The environmental samples from the Wuhan market that tested positive for SARS-CoV-2 might resolve the stalemate over the virus’ origin if they can reveal a specific animal source of the virus. “If you find a positive sample with, say, lots of raccoon dog DNA, you’ve got a hit” on the likely source of SARS-CoV-2, says evolutionary biologist David Robertson of the University of Glasgow, who co-authored the epicenter paper. But the preprint by Gao and colleagues only notes that those samples contain DNA from many species without specifying which ones—other than humans. “The authors have already done the analysis, they have just not put all the results needed to interpret them in their paper,” says evolutionary biologist Andrew Rambaut of the University of Edinburgh, a co-author of both studies. “This will undoubtedly be fixed if the paper gets through peer review.” Still, Worobey and his co-authors concede, even that evidence might not be enough to end this polarizing debate. “With the way that people have been able to just push aside any and all evidence that points away from a lab leak, I do fear that even if there were evidence from one of these samples that was full of red fox DNA and SARS-CoV-2 that people might say, ‘We still think it actually came from the handler of that red fox,’” Worobey says. j 4 MARCH 2022 • VOL 375 ISSUE 6584

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U.N. panel warns of warming’s toll and an ‘adaptation gap’ Impact of climate change will be worst for the natural world and humanity’s most vulnerable By Paul Voosen

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ver the past 70 years, humanity has made great strides on a number of metrics: increasing life expectancy, cutting hunger and disease, boosting education levels. But a prime engine of these gains—the burning of fossil fuels—now threatens to slow down global development, according to a report released this week by the United Nations’s Intergovernmental Panel on Climate Change (IPCC). With temperatures already 1.28C warmer than in preindustrial times, some ecosystems are nearing a hard limit on their ability to adapt, including warm water coral reefs, coastal wetlands and rainforests, and the frigid mountain and polar realms, the report warns. And although humanity can adapt to warming more easily than the natural world, it needs to move faster, says Michael Oppenheimer, a climate scientist at Princeton University and one of 270 report co-authors. “We’re not keeping up. The rate of climate change is faster than our ability to figure out how to deal with climate change.” The report is part of IPCC’s sixth assessment of climate science, a process its volunteer scientists undertake every 7 to 8 years. A first report, released in August 2021, documented the evidence of climate change: rising seas, extreme heat, severe storms. The new report looks at its impacts on humans and nature—and our ability to adapt to it. (A third report, on reducing emissions, is due in April.) The report recounts a familiar litany of present-day impacts. Half of the more than

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4000 plant and animal species studied have shifted poleward or to higher elevations. Corals are bleaching, forests are burning, and marine heat waves are killing swaths of species. Yet the severity of the ecosystem impacts still surprised the report’s authors, says Camille Parmesan, an ecologist at the University of Plymouth. Particularly alarming, she says, are the thawing of permafrost and drying of tropical peatlands, which are, in some years, turning these natural absorbers of carbon dioxide into emitters that could accelerate climate change. “We have an increased risk of irreversible impacts,” Parmesan says. Humans aren’t immune. Rising heat and humidity are increasing the number of days where outdoor exertion is nearly impossible and worsening pregnancy outcomes, the report finds. Disease vectors such as mosquitoes have benefited from longer warm seasons and expanding ranges. Worsening fires have increased smoke exposure and incidence of respiratory disease. “People are now suffering and dying from climate change,” says Kristie Ebi, a co-author and epidemiologist at the University of Washington, Seattle. Drought has slowed the global growth in farming productivity, needed to feed growing populations. Ocean warming and acidification have damaged fisheries and shellfish aquaculture. Storm surge and flooding, worsened by rising seas, are damaging coastal cities. Although the influence of climate on migration and human conflict is murky, severe weather is already displacing populations. “It’s a red flag for the future,” says Brian O’Neill, director of the Joint Global Change Research Institute and a report co-author. Across the board, the effects will get worse.

Even if global warming can be held to 28C by later this century—which might be feasible if nations stick to emissions pledges made last year at the U.N. climate meeting in Glasgow, U.K.—up to 3 billion people could face water scarcity. Snowmelt for irrigation could decline by 20% in many river basins; ocean saltwater could displace fresh groundwater on small islands. Food insecurity will worsen, with malnutrition increasing in the global south. Exposure to dengue fever will grow. No matter the scenario, 1 billion people will be exposed to chronic flooding from rising seas. If warming reaches 38C or higher, it’s possible that in some locations, sweating will no longer be enough to keep the human body from overheating. The Persian Gulf will be the first to reach that threshold, but “It’s going to become a problem in many places of the world, including, eventually, the United States,” Oppenheimer says. The magnitude of the effects will depend heavily on underlying social conditions such as poverty, health, and governance, the report emphasizes. For example, O’Neill notes, the number of people forced into poverty over a span of 15 years by climate change could range from 10 million to 100 million depending on their vulnerability and that of their lands. Preparing for climate change, the report concludes, is not simply a matter of building seawalls or irrigation systems. “Equally important is improving living conditions across the world,” O’Neill says. Most projects to adapt to this future are small, fragmented, and focused on near-term risks, the report finds. “There’s an adaptation gap,” Oppenheimer says. “Governments are paying much more lip service than actually doing a lot.” So far the adaptations mostly focus on water: levees and flood warning systems, coastal wetland restoration, soil moisture conservation for farming, and armoring of coastlines. Bolstering access to health care or establishing heat emergency plans would also make societies more resilient. And the report calls for adaptations to preserve the natural world: restoring the diversity of forests, aiding the migration of species, and protecting more lands and waters to give species space to adapt. Still the natural world will suffer. But for humanity, IPCC sees some hope, projecting that living conditions will continue to improve under many scenarios—just more slowly than they have in the past. “We’re trying not to jeopardize the progress we’ve made,” O’Neill says. “Though that can be hard to keep in mind when you look outside the window.” j science.org SCIENCE

PHOTO: AHMAD AL-RUBAYE/AFP/GETTY IMAGES

Increasing drought in the Middle East will jeopardize farming throughout the region.

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COVID-19

China quietly plans a pivot from ‘zero COVID’ Scientists are studying how to live with the virus while avoiding a crisis like in Hong Kong By Dennis Normile

of 7.4 million followed its own zero COVID approach that eschewed citywide lockdowns. hina’s aggressive “zero COVID” stratIt worked relatively well through December egy has served it remarkably well. 2021, but with the arrival of the Omicron The country has reported fewer than variant, cases have soared. Although vacci154,000 cases and 5200 deaths from nation coverage overall is at 76%, only 46% COVID-19 so far. But as the highly of people in their 70s and 29% of those in transmissible Omicron variant their 80s were fully vaccinated. Many elderly seeps into the country and the social and were alarmed by early reports of side effects economic costs of the zero COVID policy and felt confident in the city’s ability to keep mount, Chinese researchers are examining the virus at bay. Deaths are concentrated options for coexisting with the virus, as the among those who shunned vaccination, says rest of the world is doing. Some think that virologist Jin Dong-Yan of the University of shift may soon begin. Hong Kong (HKU). Unless stricter measures It will be a momentous decision, and the are introduced, 4.6 million Hong Kong resicountry is sure to proceed cautiously. China dents will have been infected by mid-May, wants to avoid COVID-19 outmodeling by HKU researchers breaks like the one now oversuggests. More than 3200 will whelming Hong Kong, which have died. reported more than 34,000 China, too, will face a wave of new COVID-19 infections and infections during any transition. 87 deaths on 28 February alone. In places that lack community Models predict that toll will health clinics or general pracclimb further. titioners, even those with mild Yanzhong Huang, a global symptoms are likely to rush to health specialist at the Counhospitals, and “medical resources cil on Foreign Relations, a U.S. will quickly be exhausted,” Xi think tank, says until recently he says. Although the vaccination believed China might introduce rate now tops 87%, and more more flexible measures as early than 550 million people have as this month. Now, “It is very received boosters, vaccination of likely that Chinese leaders may the elderly lags, especially in ruwait till the dust settles” from ral areas. And with many Chinese the Hong Kong crisis, he says. Xi vaccines relying on inactivated Chen, a public health scientist at A temporary isolation facility for COVID-19 patients in Hong Kong, where cases virus rather than the messenger the Yale School of Public Health, and deaths have soared since December 2021. RNA technology common in the says China needs more time—up West, it’s unclear how fast their to 1 year—to further raise vaccination and outweigh the benefits,” says Zhangkai Cheng, protection wanes or how well they will fare booster coverage and bolster rural health a respiratory specialist at Guangzhou Mediagainst new variants, says immunologist care capabilities. cal University. “Whether that point has arRustom Antia of Emory University. China’s zero COVID policy has relied on rived is up for debate.” Given the high stakes, many predict Chimass testing, contact tracing, isolating the The national government is already pushna’s leaders will proceed cautiously. Huang infected, restrictions on international and ing back at what it considers unnecessary loenvisions steps such as reducing the length of domestic travel, and lockdowns of entire cal restrictions. On 18 February, the National quarantines and putting fewer contacts into cities. The system has helped China stamp Development and Reform Commission told isolation. Yale’s Chen thinks China might first out every outbreak so far, including several local governments to avoid arbitrary lockopen up one city or region as a test case. of the Omicron variant. But outbreaks are downs and barred unauthorized closures of China’s big leap may affect the rest of the becoming more frequent and widespread. restaurants, supermarkets, tourist sites, and world as well. Unleashing COVID-19 on a On 25 February, the National Health Comcinemas. The Chinese Center for Disease population of 1.4 billion means a lot of peomission reported 93 confirmed cases of loControl and Prevention is studying changes ple “will be brewing the virus,” says Gabriel cal transmission in 10 provinces, despite to existing control measures to “ensure norLeung, HKU’s dean of medicine. That will the burdensome countermeasures. Shenmal international exchanges and economic provide ample opportunity for new variants zhen, which borders Hong Kong, recently development,” its chief epidemiologist, to emerge. “It’s not just a national problem, closed museums, libraries, many parks, and Zunyou Wu, said on 15 February. it’s actually a global issue,” Leung says. j beaches in response to an uptick in cases. But the situation in Hong Kong shows why Apartment compounds face lockdowns if caution is needed. The semiautonomous city With reporting by Bian Huihui.

PHOTO: ANTHONY KWAN/GETTY IMAGES

C

SCIENCE science.org

even one resident tests positive. Most people must get tested every 48 hours. “The huge inconveniences and difficulties imposed upon people’s livelihoods and lifestyles may be turning the wheels of the Chinese policy machinery to consider some kind of policy adjustment,” China political analyst Chen Gang of the National University of Singapore (NUS) wrote in a February commentary for Channel News Asia. And COVID-19 countermeasures started to dent China’s economic growth in the second half of 2021, says Xi Lu, an NUS specialist in Chinese economic policy. “All of the economic indices point to a continued decline,” Xi says. “There will likely come a point when the costs [of zero COVID]

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By Jeffrey Mervis; Graphics by Kelly Franklin

A rising tide, but not for Black students (1)

Black majors rare at most U.S. campuses (2)

The number of U.S. undergraduate degrees in physics had tripled by 2020 compared with 1999. Although the absolute number of Black physics majors also rose, their share of the total plummeted.

Some one-third of all U.S. physics departments graduated no Black physics majors between 1999 and 2020. Despite their small size, historically Black colleges and universities (HBCUs) far outpaced predominantly white institutions in awarding bachelor’s degrees in physics to Black students.

CAN U.S. PHYSICS

RECORD OF EXCLUSION?

Physics bachelor’s degrees awarded

Data show white institutions falling short and Black colleges leading the way

9000 Black students

Other students

0 BLACK MAJORS

1–2

One institution HBCU

6

3

3–4

0 1999

2005

2010

Black bachelor’s degrees 300

2015

2020

Black bachelor’s degrees (%) 262

5 4.8% 4

2 155

3.1%

3 2

1

1 0 1999

950

2005

2010

2015

2020

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0 1999

2005 2010

2015 2020

260 institutions

248

140 science.org SCIENCE

CREDITS: (ILLUSTRATION) C. SMITH/SCIENCE; (DATA) NATIONAL CENTER FOR EDUCATION STATISTICS/INTEGRATED POSTSECONDARY EDUCATION DATA SYSTEM/COMPLETIONS 1999–2020 (2020 DATA ARE PROVISIONAL)

I OVERCOME ITS

n the 1990s, physics departments at U.S. universities faced an existential crisis. The number of undergraduate physics majors had plummeted by 25% over 10 years, prompting fears that many departments might disappear or be merged into other programs. Academic physicists scrambled for ways to attract more students, making lectures more interactive and updating the curriculum. The changes had the desired effect: The annual number of physics majors, the first rung in the career ladder, nearly tripled over the next 2 decades (see graphic 1). Black students, however, were left behind. In 2017, the American Institute of Physics (AIP) assembled a National Task Force to Elevate African American Representation in Undergraduate Physics & Astronomy (TEAM-UP), the discipline’s first deep dive into the lack of diversity at the undergraduate level. The task force’s 2020 report documented the crisis in dispiriting detail. Data from the U.S. Department of Education show the percentage of undergraduate degrees awarded to Black students dropped from 4.8% in 1999 to 3.1% in 2020. Had the number of Black undergraduates earning physics degrees simply kept pace with the overall growth in the major, the current annual total would exceed 350. Instead, it was 262 in 2020. As bad as those numbers are, they hide how rare Black students are on most U.S. campuses. Some 30% of the 853 U.S. departments awarding physics degrees did not graduate a single Black student between 1999 and 2020, and an additional 30% graduated just one or two (see graphic 2). In contrast, historically Black colleges and

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universities (HBCUs) lead the nation in graduating Black physics majors despite their relatively small size and limited resources. The demographics at the graduate level are even more depressing. Black students made up less than 1% of Ph.D. recipients in physics in 2019 (see graphic 4). Over the past 2 decades, the absolute number of physics Ph.D.s awarded to Black students each year has remained essentially flat. In contrast, the number awarded to Hispanic students has increased substantially (see graphic 3). The near invisibility of Black people has made it easier for the physics community to ignore their perspective. This special package, which draws from interviews of more than 50 Black scientists, seeks to rectify that situation and amplify their voices. They tell of battling systemic racism, which creates an uneven playing field, as well as pushing back against a culture that continues to question their ability and desire to do physics. They describe how that dominant culture in academic physics expects Black people to lead the way in correcting a problem not of their making. They talk about struggling to gain the same professional recognition, respect, and rewards that accrue to white physicists, and how working to improve diversity can jeopardize their own scientific careers. They also describe isolation and debilitating self-doubt, and their frustration and anger that decades of rhetorical support for improving diversity haven’t translated into better demographics. Especially poignant are the experiences of those who have broken through the double barriers of race and gender. In 1972, Willie Hobbs Moore became the first Black woman to earn a U.S. doctoral degree in physics. Exactly 50 years later, the total stands at only 150, according

to a list (at aawip.org) compiled by physicist Jami Valentine Miller, a senior examiner with the U.S. Patent and Trademark Office. In 2007, she became the first Black woman to earn a Ph.D. in astrophysics from Johns Hopkins University. Many U.S. graduate physics departments have never handed a diploma to a Black woman. Although Black scientists in other disciplines face similar barriers, this package examines physics because, as the TEAM-UP report notes, the community’s record on diversity “is particularly depressing.” The stories describe how the “priesthood,” the white men who constitute a majority in the field, has stymied efforts to make meaningful change through a sense of white privilege (see p. 952), and how some universities are building a more welcoming environment by dismantling policies that disadvantage students from marginalized groups (see p. 956). One story examines the outsize role HBCUs have played in educating Black physics undergraduates, and how that role is threatened (see p. 960). Another shows how a graduate program at an elite, predominantly white institution has been more successful than its peers in training black Ph.D.s, using a model that was not intended to address diversity at all (see p. 967). The package also explores the disproportionate number of Black Ph.D. physicists who are drawn to teaching at the precollege and community college levels (see p. 964). Together, these stories attempt to portray the Black experience in U.S. physics and identify institutions and programs that offer models for change. But there are no simple prescriptions. As the TEAM-UP report emphasizes, the physics community must first internalize how it got to this dismal point before it can make meaningful improvements in the culture. j

Growing gap with Hispanic students (3)

A dearth of Ph.D.s (4)

The number of U.S. physics Ph.D.s awarded to Hispanic students has tripled over the past 2 decades while remaining essentially flat for Black students.

Number of Ph.D.s awarded

60

Black

Black students are underrepresented by a factor of 10 in U.S. doctoral physics programs, which train mainly domestic white students and those from other countries.

60

Hispanic

50 40 30 20

White 41.3%

Black 0.5% Multiethnic 1.5% Hispanic 2.7% American Indian 0.1% Hawaiian/Pacific Islander 0.05%

Foreign 44.3%

Unknown 4.4%

19

16 10

Asian 5.2%

7

0 1999

2005

2010

2015

2020

The top 10 producers of Black physics majors are HBCUs (1999–2020)

5–7

1. Morehouse College 188 2. Xavier University of Louisiana 142

3. Dillard University 92 4. Benedict College 84 5. Alabama A&M University 69

6. Hampton University 60 7. Tuskegee University 58 8. Lincoln University 58

9. Jackson State University 57 10. Spelman College 56

8–10 11–15

85

44 SCIENCE science.org

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16–25

21

26–50

13

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Physicist Apriel Hodari has documented how the views of a white male “priesthood” shape the culture of U.S. physics.

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THE TOLL OF WHITE

PRIVILEGE How the dominant culture in physics has discouraged diversity By Jeffrey Mervis

CREDITS: (ILLUSTRATION) C. SMITH/SCIENCE; (PHOTO, OPPOSITE PAGE) EMAN MOHAMMED

A

priel Hodari has spent many years studying how to improve training and reduce inequity in the scientific workforce. That research has brought her face to face with “the priesthood,” her name for the dominant white male culture in U.S. physics. White men, for example, hold 70% of the faculty positions at U.S. universities that grant physics Ph.D.s. Hodari wondered whether there was a connection between the priesthood’s views on issues such as racism and sexism and the profession’s deplorable record on diversity. So in 2017, Hodari, a senior scientist at a California company that helps researchers obtain federal grants, and Melissa Dancy, a physics educator at the University of Colorado, Boulder, received support from the National Science Foundation to interview 27 white, male, academic physicists, from senior professors to graduate students. What the researchers heard during those conversations didn’t surprise Hodari, who is one of only 150 Black women to earn a physics Ph.D. in the United States over the past 50 years. “Not me. Not my colleagues. Not my time. Not my field,” is how Dancy, who is white, summarized their thoughts on why U.S. physics lacks diversity during a presentation last year to a meeting of science education researchers. Most of those in the small sample—chosen from those who volunteered to participate— voiced concerns about equity and diversity, Hodari and Dancy found. But they were SCIENCE science.org

skeptical that racism and sexism were prevalent in physics. They also couldn’t think of any instances of racist or sexist behavior in their classrooms or labs. And to a man, they believed they were in no position to ease the harm such biases might have on the profession. “In other words, [any problem] is very far away from me,” Dancy reported, “and I have no responsibility for it.” Hodari and Dancy believe their interviews also offer a peek into the corrosive impact of white privilege on physics. Scholars coined the term decades ago to describe how white people use their membership in a dominant group to assert political, cultural, and economic power over those outside that group. White privilege allows physicists to “not pay attention to systemic racism because there aren’t any consequences,” Hodari says. She thinks it also explains why many of her colleagues “are completely clueless” about the harmful effects on their profession of attitudes and actions born of privilege and racism. Black physicists don’t have the luxury of ignoring white privilege. Every one of the more than 50 Black scientists interviewed for this special package described experiences, beginning in their student years and extending into senior faculty positions, in which they were confronted with pernicious behavior associated with white privilege. Some episodes were blatantly racist—white people mocking their appearance or denigrating their intellect— whereas others were more subtle, such as

comments based on negative assumptions about their backgrounds or reasons for wanting to be a scientist. Battling white privilege requires constant vigilance, and it can take a heavy toll. “I didn’t choose physics to clean up a mess I didn’t make … [but] I’m bleeding so that people can learn about this toxic environment we face,” says Jessica Esquivel, an associate scientist at Fermi National Accelerator Laboratory (Fermilab) and an advocate for social justice and greater opportunities for Black girls in science. Esquivel, who identifies as an Afro-Latinx lesbian, says she’d rather be recognized for her work on the properties of the muon or neutrino physics. But her activism, she says ruefully, is often “the only thing [my colleagues] notice about me.” White privilege must be fought on the individual and institutional levels, says Marta McNeese, chair of the physics department at Spelman College, an all-women historically Black college in Atlanta. “Removing barriers to participation isn’t enough,” says McNeese, who is Black. “If you have someone in your department who is toxic, you need to figure out how to protect students from them.” ONE DAMAGING consequence of white privi-

lege is what’s known as “the Black tax.” It stems from an assumption that Black faculty members, because they are the victims of racism, should do more than their white colleagues to promote diversity. That means spending more time recruiting and mentoring students from underrepresented groups, 4 MARCH 2022 • VOL 375 ISSUE 6584

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Physicist Jessica Esquivel, who works on the Muon g-2 experiment at Fermi National Accelerator Laboratory, says battling white privilege can take a heavy toll.

basket—and one carried by a Black man. “What if for some reason I had to leave?” Nord says. “Why not build upon the work of the entire cohort of Black scientists at the lab?” Nord is also worried about what happens if the lab’s demographics don’t improve. “Does it then become my fault?” LOCKYER WON’T be around to see whether his

strategy of asking Nord to lead Fermilab’s diversity effort succeeds, having announced his retirement in fall of 2021. But a similar effort at Stanford University 5 decades ago suggests expecting one Black leader to shoulder the burden can falter without sustained institutional support. In the 1970s, Stanford administrators tacitly agreed to let Arthur Walker, a solar physicist recruited in 1974 as the first Black member of the department, take on the task of diversifying both graduate enrollment and the faculty ranks. “Art was a top scientist, and he brought in others [Black students and faculty],” recalls physicist Arthur Bienenstock, a white emeritus professor and special assistant to the Stanford president who served as the university’s first affirmative action officer in the early 1970s. “Looking back, I can’t say that I or anyone else did anything that was really effective.” Walker, however, had an impact. In fact, Stanford often claimed that, from the early 1970s to 2000, it led the nation in awarding physics Ph.D.s to Black students. (The total is believed to be roughly two to three dozen, but Science could not confirm that because of

a lack of documentation.) The flow of Black graduate students had already begun to dry up, however, by the time Walker died of cancer in 2001. And since his death, Stanford’s track record is indistinguishable from that of other elite graduate physics programs. Current and former white Stanford professors say one reason for the drop-off was their failure to recruit Black students. Robert Wagoner, who retired in 2012 after 40 years on the physics faculty, recalls “edicts” from department chairs to recruit at conferences they attended that attracted large numbers of students from marginalized groups. But Wagoner always came away empty-handed. “Everybody wanted them,” he says about the students Stanford tried to recruit. “We did all we could. We’d call them and encourage them to come. But the pool was so tiny.” Such explanations infuriate Roscoe Giles, a computer engineer at Boston University and longtime diversity advocate who in 1975 became the first Black student to earn a Stanford physics Ph.D. “It drives me crazy,” he says, “to hear people from elite places like Stanford say, ‘We can’t compete.’ It’s the opposite of what you hear them say the rest of the time about their ability to attract the most talented faculty and students.” OTHER BLACK PHYSICISTS told Science they

have had difficulty gaining institutional support for efforts to increase diversity— even when they volunteered to lead them. Physicist Kim Lewis, for example, says she worked hard to attract Black students and science.org SCIENCE

PHOTO: FERMI NATIONAL ACCELERATOR LABORATORY

more service on diversity committees, and more community outreach. The tax carries a double penalty because those extra efforts— which many Black physicists take on willingly and without compensation—are often discounted, if not ignored, when physicists come up for tenure and promotions. Physicist Adrienne Stiff-Roberts started to pay that tax in 2004 after coming to Duke University in North Carolina, where she says she became the “first—and still only” Black member of the physics department. The North Carolina native says she “knew that Duke had a reputation for not being a welcoming place for Black people,” a history that university officials have acknowledged. But she accepted the tenure-track position with the hope of improving the situation. As Stiff-Roberts moved up the academic ladder, she poured her energy into fostering greater diversity by training graduate students, mentoring undergraduates, and running a Saturday morning academy for middle-school students. “You can get sucked into trying to make your institution a better place,” she says. But she also kept track of the toll such activities were taking on her research and turned down invitations that didn’t feel genuine. “Don’t ask me to do a lot of work that you don’t value,” Stiff-Roberts says. Instead, she says, “I decided that the best thing I can do is to succeed and become an existence proof.” Computational cosmologist Brian Nord faces similar challenges at Fermilab, the U.S. government’s premier particle physics center. Along with Esquivel, Nord is part of a group of five Black lab employees who issued a 17-page “Change-Now” manifesto in June 2020, just days after Minneapolis police officer Derek Chauvin murdered George Floyd. In addition to demanding that Fermilab officials hire and retain more Black scientists and adopt policies to achieve social justice, the manifesto urged them to “listen to and do what Black employees say they need, and not make plans for us without us.” Several months later, Fermilab’s director, Nigel Lockyer, who is white, asked Nord to lead efforts to hire and promote more minority scientists. “I want to build around Brian,” Lockyer said about Nord, who had recently been promoted to become the lab’s only tenured Black scientist. “I need somebody to be an attractor, if you will, so that a young Black scientist [looking for a job] would say, ‘Gee, I want to work with Brian.’” The 39-year-old Nord says he is honored to receive such a vote of confidence and believes he has “demonstrated that I can lead on these issues.” But he thinks Lockyer’s approach falls far short of the necessary institutional commitment. In particular, Nord wonders why Lockyer decided to put all his eggs into one

faculty to the Rensselaer Polytechnic Institute (RPI), a predominantly white institution in upstate New York where she was a faculty member between 2006 and 2017. But she says senior administrators often pushed back against her efforts. “I had been doing a lot of recruiting, on my own time,” Lewis says, “and one day I realized that none of these students looked like me.” Changing the demographics would require RPI to commit time and money, she concluded. “But whenever I tried to bring it up, the response was, ‘We shouldn’t need to make an extra effort to attract them. They should want to come. We’re RPI.’” In 2018, Lewis moved to Howard University, a historically Black college in Washington, D.C., where she is now a professor of physics and associate vice president of research. RPI’s attitude toward recruiting a more diverse student body wasn’t the only reason for the move, she says. Another factor was the way she was treated by RPI undergraduates. “The students there can be brutal,” she says. “They didn’t like my hairstyle, they accused me of speaking Ebonics [rather than English], and they said I looked like a monkey standing in front of the class.” In contrast, she says, “At Howard I can take off my dean’s hat and, for a few minutes, be an aunt,” alluding to her approach to mentoring. “I couldn’t do that at RPI.” Lewis says she chose not to file a formal complaint at RPI because, as a petite Black woman, she feared for her safety. “I have to walk around campus, and I just don’t trust [the students],” she says she told a university official at the time. RPI declined requests from Science to address the issues Lewis raised. The arrogance Lewis says she encountered at RPI is a common manifestation of white privilege in physics, Hodari says. “It’s hard to empathize with the problems of marginalized populations when you think you’re the smartest person in the room and have all the answers,” she says. Such smugness also hinders efforts to improve diversity, says Mary James, a physicist at Reed College and co-chair of an American Institute of Physics task force that issued a 2020 report on how to reverse the declining percentage of Black undergraduates majoring in physics. “It’s hard for some physicists who think they are good at everything to say to themselves: ‘Maybe what I’ve been doing for so long hasn’t been effective,’” she says. Another impediment to change, says James, who is Black, is that physicists can harbor condescending attitudes toward advice from experts in other fields. For example, physicists who pride themselves on making SCIENCE science.org

decisions based on “hard data” may scoff at survey data from social scientists in which students describe deficiencies in their training, she says. “But qualitative data are data, too,” James says. She and others think social scientists could help physicists address cultural, sociological, and institutional issues that might sabotage diversity initiatives. EVEN MEMBERS of the priesthood who have

embraced the value of diversity say change doesn’t come easily. Keith Bechtol, a white cosmologist at the University of Wisconsin (UW), Madison, says he barely noticed the absence of Black students among the 18 physics majors in his graduating class at the College of William and Mary in 2007. And Bechtol thought little about having “maybe one Black man” among the 45 students in his graduate program at Stanford, where he earned his Ph.D. in 2012.

“

If you have someone in your department WHO IS TOXIC, you need to figure out how to protect students from them. MARTA MCNEESE, SPELMAN COLLEGE

What finally opened his eyes to the corrosive effect of white privilege, he says, was his first teaching assignment at UW as an assistant professor in 2018. After asking students in his introductory physics class to write a paper on a physics Nobel laureate, he says, “I realized that I was perpetuating all these inequities by forcing them to write almost exclusively about white males.” Bechtol revised the assignment to allow students to write about any physicist. Then he began to devote one class period in the course to a discussion of diversity, equity, and inclusion. Last year, he won a departmental grant to fund research fellowships for a handful of undergraduates from groups traditionally underrepresented in physics. He’s hoping other faculty members will sign up to mentor those students, leading

to a more welcoming environment at UW. “We’re not there yet,” he admits. The toptier research institution has no Black faculty in its 48-member physics department, he notes, and records show only two Black students earned undergraduate physics degrees in the 5 years from 2015 to 2019. Advocates for greater diversity say many more white male physicists will have to follow Bechtol’s lead if the field wants to reverse the declining participation by Black students. And Nord says the first step is a real commitment to change. “At every place I’ve worked, I’ve witnessed promises unkept and seen racism in their committees,” Nord told the leaders of the American Physical Society last year during a webinar on the value of diversity. “Most of my secure, senior colleagues—white men— encourage gradualism, and whitesplain to me that ‘change takes time.’” Any change must also be accompanied by greater accountability, Nord says. “Most of the talk I hear about helping people of color navigate the treacherous waters [in physics] amounts to suggestions on how to hop from one lily pad to another to avoid falling into deep water,” Nord says. “But that’s not enough. You also need to take the negative players out of the game by identifying and enforcing consequences for bad behavior.” Those consequences, he says, could include preventing scientists and institutions from receiving federal research grants if they can’t show progress in improving diversity, equity, and inclusion on their campuses. Other diversity advocates, however, think such a punitive policy would be counterproductive. They want the federal government and other funders to use the carrot rather than the stick, offering additional support to institutions and departments that are doing the right thing. Either way, diversity advocates agree that lasting change won’t happen until the priesthood becomes fully engaged. Nadya Mason, a professor at the University of Illinois, Urbana-Champaign, says she is still waiting for that to happen. Mason, who is Black, recalls a senior white faculty member asking her for advice during a 2020 campus event on how to combat racism. “I don’t know what to do. Can you help me?” he asked her. “I told him that the point of the event was for whites to think about their actions,” Mason says, “not to ask Black people what they think. I don’t think he liked my answer. But until white people educate themselves, nothing will happen.” j 4 MARCH 2022 • VOL 375 ISSUE 6584

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FIX THE SYSTEM,

NOT THE STUDENTS

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hysicist Mary James was a sophomore at Hampshire College in Massachusetts in 1974 when a professor encouraged her to apply for a prestigious internship at a world-class laboratory. Such competitive internships are often an essential step to becoming an academic scientist, which was her goal. But for a young Black woman from Chicago, the idea of spending 10 weeks at what is now called the SLAC National Linear Accelerator Laboratory in California seemed far-fetched. “I had never been west of the Mississippi,” she says. “And the brochure was so intimidating that I thought, ‘This isn’t even worth a stamp,’” James recalls. But her Hampshire professor kept pushing her 956

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By Jeffrey Mervis

to apply, and the next year she did—and was accepted. Spending two summers at SLAC set her on the road to earning a Ph.D. from Stanford University and then to Reed College, where she’s been a faculty member for 35 years. Nearly a half-century later, James can still recall a lunch at SLAC that cemented her future. “A bunch of us students were sitting at the grown-ups’ table,” she says, “eavesdropping on a very lively conversation, when I had an epiphany: ‘They are getting paid to do this.’ I hadn’t realized you could make a living doing physics.” James also didn’t realize that the internships were making her more acceptable to the white male “priesthood” that sets the discipline’s culture (see p. 952). That culture, which has historically excluded people

who look like James, expects students to meet a certain standard. And rather than giving students the help they need, James and other Black physicists say, the priesthood too often decides that those students are unworthy of joining the profession. That way of thinking is so common in physics that it even has a name. Diversity scholars call it the deficit model, and they say it’s a major reason fewer than 4% of all undergraduate physics degrees awarded by U.S. institutions go to Black men and women. Sadly, that underrepresentation is getting worse: A 2020 report by the American Institute of Physics (AIP) found Black people now comprise a smaller slice of those degrees than they did 2 decades ago. James was co-chair of AIP’s National Task Force to Elevate African American Represenscience.org SCIENCE

CREDITS: (PHOTOS) REED COLLEGE; (ILLUSTRATION) C. SMITH/SCIENCE

Change requires building bridges, removing barriers

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Reed College physicist Mary James co-chaired a 2020 American Institute of Physics report on improving recruitment and retention of Black physics majors.

tation in Undergraduate Physics & Astronomy (TEAM-UP), which wrote the report. It calls on physicists to reject the deficit model and replace it with something that assigns them primary responsibility for diversifying their profession. Black students “have the same drive, motivation, intellect, and capability to obtain physics and astronomy degrees as students of other races and ethnicities,” the report says. “[But] they are choosing majors that are perceived as being more supportive and/ or rewarding.” “Black students don’t need to be fixed,” James says, driving home the point. “We know that there are a robust number of African American students capable of doing physics because they are majoring in other STEM [science, technology, engineering, and math] fields. So instead of trying to change them, let’s talk about why they are turned off by the environment in physics and work to change that.”

PHOTO: REED COLLEGE

THE TEAM-UP REPORT recommends several

ways to improve that environment. One is for departments to help students develop what it calls a “physics identity”—the ability to see themselves as a future member of the profession. Another is for physics professors to abandon their traditional role as gatekeepers who only let the “best” students advance and instead find ways to attract and retain a more diverse group of students. Students at historically Black colleges and universities (HBCUs, see p. 960) take that kind of nurturing for granted, says SCIENCE science.org

Arlisa Richardson, who also served on the TEAM-UP task force. Richardson is a physics professor at Chandler-Gilbert Community College and an alumna of Grambling State University, an HBCU in Louisiana. “At Grambling, people were rooting for us and helping us deal with any problems,” says Richardson, who nearly dropped out of physics during graduate school at a predominantly white institution because of what she and a small cohort of other Black students regarded as a hostile learning environment. Richardson has spent the past decade at Chandler trying “to re-create the experience I had at Grambling,” she says, “by providing students with a welcoming environment, hands-on learning, clubs that are student-led, and free tutoring.” Even before the TEAM-UP report, some academic physicists had begun to take similar steps. At Vanderbilt University, astrophysicist Keivan Stassun was moved to act by data showing Black undergraduates are being pushed out of the field they want to study. “The percentage of incoming freshmen who express interest in astronomy [1%] is not different between majority and minority students,” says Stassun, who identifies as Hispanic and says he has “lived the life” of someone from an underrepresented group in science. “But 2 years later, 60% of all students have fallen away, while the rate is 90% for Black students. It’s a massive disparity from which we never recover.” Similar attrition is occurring across the country, according to a recent decadal study

for astronomy and astrophysics by the National Academies of Sciences, Engineering, and Medicine (NASEM), in which Stassun took part. It reports that, among undergraduate students who say they intend to major in physics or astronomy, those from groups underrepresented in science are almost three times less likely to complete the degree than are white students. Studies have shown research internships like the one James did at SLAC can help keep students engaged in physics. But some students who would benefit most from such an experience can’t participate, argues Sheila Kannappan, an astrophysicist at the University of North Carolina (UNC), Chapel Hill. Kannappan identifies as a woman of color—“I’m half-Indian, and I appear vaguely brown.” She is also the department’s associate chair for diversity, and once ran summer research internships at UNC funded by the National Science Foundation (NSF). But after several years, she realized NSF’s research experiences for undergraduates (REU) program wasn’t a great fit for her target audience of students from groups underrepresented in science. “You had to be a superstar to get in,” Kannappan says. But many of the students she wanted to serve “don’t look good on paper—they don’t have 4.0 [grade point averages] and previous research experience. They may also need to work during the summer, or if they are transfer students from a community college, they may need to go to summer school” to catch up on prerequisite courses for their major. Instead of trying to win another REU grant, Kannappan decided to create a 2-week paid boot camp just for UNC students, including those from marginalized groups. It teaches them computational skills applicable to any area of physics, and is supported by institutional funding and a supplement to her standard NSF research grant. Its short duration, she says, “still allows [students] to go home or take [other] classes.” In fall of 2021, Kannappan also helped stand up a new peer mentoring program that pairs UNC graduate students in physics with undergraduate students in the department. “We understand that there are other things besides their coursework that affect someone’s ability to succeed in school,” says Zack Hall, who co-directs the program. All of the participants in the inaugural class—six mentees and five mentors—are from groups underrepresented in physics, says Hall, who is on track to become the first Black student to earn a physics Ph.D. from the university. 4 MARCH 2022 • VOL 375 ISSUE 6584

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The program relies on volunteer mentors, and Hall says he’s careful to make sure the 10 hours or so a week he spends coordinating their activities doesn’t interfere with his work on lattice quantum chromodynamics. But Julieta Gruszko, a white assistant professor of physics at UNC, thinks such mentors should be compensated in order to recognize their value to strengthening the profession. So in 2020, when she negotiated her startup package, she included funding for a graduate student to work with individual students and organize public events to promote diversity. Kannappan and Hall hope these and other efforts will ultimately help UNC improve its mediocre record of training Black physicists. Black students earned just seven of the 232 undergraduate physics degrees it awarded over the past decade, a rate slightly below the national average. Similar efforts are still uncommon at other campuses around the United States, according a 2020 AIP survey of 310 physics department chairs. Three out of four chairs identified “low enrollment or retention of historically underrepresented groups” as a major challenge. However, barely half of the department chairs listed “creating an inclusive learning environment”—exactly what the TEAMUP report says is essential for improving retention rates and increasing diversity— as something they need to address. And fewer than two in five identified improving the department’s “climate” as a priority. 958

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PRODUCING MORE Black majors at the under-

graduate level is only the first step in diversifying the profession. Such efforts will be for naught if they aren’t sustained. Graduate training at predominantly white institutions poses its own set of challenges, because physics departments historically have only recruited from a small and homogeneous group of undergraduate schools and use entrance requirements that often put Black students at a disadvantage. One increasingly popular alternative is for those departments to partner with institutions that serve large numbers of students from minority groups. Those arrangements, often called bridge programs, come in many flavors. But they share the goal of increasing the number of Black students earning graduate degrees in the natural sciences. Simultaneously, many departments have broadened their definition of a viable candidate. In 2004, Stassun decided to combine those elements into a bridge program that has become a national model. Students apply to a master’s degree program at Fisk University, an HBCU also located in Nashville, Tennessee, that feeds into a doctoral program at Vanderbilt. It caters to promising students with holes in their undergraduate education that need to be filled before they can begin a research-based doctoral training program. Simultaneously, Vanderbilt reduced what Stassun calls an “overreliance on standardized test scores,” in particular

the graduate record examination (GRE) in physics, to winnow the initial applicant pool. Studies have shown GRE scores are poor predictors of success in graduate school, and many minority students who are otherwise qualified do poorly on the exam, Stassun told a meeting last year of the Roundtable on Black Men and Women in Science, Engineering, and Medicine sponsored by NASEM. The Fisk-Vanderbilt program puts more weight on factors such as perseverance, the ability to set long-term goals, leadership, and community engagement. Those qualities, Stassun says, don’t penalize students of color and are also seen as better metrics for success. Using those criteria helped Vanderbilt attract a more diverse pool of students. But that was only the first step toward producing more Black Ph.D.s in the natural sciences, Stassun explains. “The next challenge was building a sense of community.” Vanderbilt’s partnership with Fisk serves that role, he says, by giving minority students “1 to 3 years to capitalize on what Fisk has to offer.” And once students began their doctoral work at Vanderbilt, Stassun took on a third challenge: creating a more hospitable environment. That required creating mentoring networks that went beyond the traditional mentoring “dyad” between a graduate student and their adviser, he says. “I can’t be their only source of support,” Stassun says. He says the broader mentorscience.org SCIENCE

PHOTO: JON GARDINER/UNIVERSITY OF NORTH CAROLINA

University of North Carolina, Chapel Hill, physics professor Sheila Kannappan (second from left) is flanked by graduate student Zack Hall (left), undergraduate Nathnael Kahassai (second from right), and graduate student Derrick Carr.

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ing networks, which include faculty from other departments and peers, allow students to tap into “all the experiences they will need” to succeed. The bridge program has made impressive strides in increasing diversity: Some 80% of the 150 students who have earned master’s and doctoral degrees identify as either Black or Hispanic, according to data compiled by the program, and more than half are women. Along the way, says Stassun, who stepped down as director of the program in 2015, Fisk has become the nation’s top producer of Black students with master’s degrees in physics and materials science. “And my lab has awarded more Ph.D.s. in astrophysics to African Americans than any other lab in the country,” he says. THE FISK-VANDERBILT bridge pro-

gram has shown that pairing a minority-serving and a predominantly white institution can boost the flow of Black students into physics. A half-dozen major research universities have adopted variations of the model, and the American Physical Society is using the concept to build a nationwide network of graduate programs that have pledged to train more minority students. (Hall earned his master’s degree at California State University, Long Beach, under one such program, which he says “really bolstered my confidence.”) But not every pairing has been successful. For example, the checkered history of the former Center for Integrated Space Weather Modeling (CISM) based at Boston University shows what can go wrong. In addition to advancing research in the emerging field of space weather, CISM hoped to alter the field’s overwhelmingly white demographics. So the center partnered with Alabama A&M University (AAMU), an HBCU with a new graduate program in space physics. The plan was to have students earn a master’s degree from AAMU before pursuing doctoral work at that university or elsewhere. The center received 10 years of funding from NSF, which wanted to foster greater diversity in the discipline. But AAMU’s program only graduated two Black students—Fana Mulu-Moore and Samaiyah Farid—over its decadelong affiliation with the center. And both women say the partnership was little help to their careers. In particular, they viewed themselves as tokens in a program they say only gave lip service to diversity.

“Sometimes it felt that they were just checking a box,” says Mulu-Moore, who spent 10 years trying to find her place in physics after also earning her Ph.D. from AAMU in 2009. “We were doing a lot of outreach to minority schools for CISM. But I don’t know of a single minority student who came through the CISM program and went into the field.” Farid left the AAMU program after her master’s degree to work at the HarvardSmithsonian Center for Astrophysics. She hoped it would be a springboard into space physics. But she says she was the only Black scientist there and was subject to racial taunts. After leaving that job and enrolling in a doctoral program at the Uni-

“

nitely stigmatizes the students,” Farid says about the Fisk-Vanderbilt bridge. “People assumed I was in the program because I was Black. And if you’re in the program, people also assume that you’re not up to par.” At the same time, her affiliation with the program has helped advance her career. She is now doing a postdoctoral fellowship at Yale University that is only available to astrophysics students who have gone through bridge programs at Vanderbilt, Columbia, and Ohio State universities. Mulu-Moore now teaches at Aims Community College in Colorado. Last summer, after several years living a precarious existence as an adjunct, she was hired as the department’s only full-time faculty member. She relishes teaching first-generation college students who remind her of herself when she arrived in the United States 20 years ago from Ethiopia. “It’s a small, nurturing school, and I feel that I’ve found my niche,” she says. Along with engaging in extensive community outreach to broaden the pool, Mulu-Moore tries to connect her most promising students with the kind of high-quality summer internships that helped launch James’s career. One of those students is Carter Woodson, a biracial student who graduated from a rural West Virginia high school and has spent 10 years trying to acquire a college education, the cost of which is often beyond his means. Woodson teaches and tutors at a local high school that Aims operates, a job that pays the tuition for his science courses and feeds his long-term ambition of becoming a science and math teacher. When Mulu-Moore told him about a 10-week, paid summer program run by the National Solar Observatory in conjunction with the University of Colorado, Boulder, he jumped at the chance. “I’ve always loved math,” he says, “and this sounds like the chance to do something really interesting.” Mulu-Moore knows he’ll face stiff competition. “These internship programs are usually looking for students from the top universities,” she says. “But Carter checks all the boxes. He’s very smart, he’s a fulltime student, and he’s absolutely determined to get a degree.” To Mulu-Moore, Woodson is exactly the kind of student the U.S. physics community should be encouraging and welcoming. “He’s just as capable,” she says, “as somebody from an Ivy League school.” j

Black students don’t need to be fixed. … So instead of trying to change them, let’s talk about WHY THEY ARE TURNED OFF by the environment in physics and work to change that.

SCIENCE science.org

MARY JAMES, REED COLLEGE

versity of New Hampshire (UNH), Farid said she experienced a similar sense of isolation and hostility toward her as a Black person. It was a “horrible experience,” she recalls. Although the department was a partner in CISM, Farid felt it offered her no additional support or guidance. But Farid didn’t give up. After dropping out of UNH and taking a short respite, she resumed her doctoral studies at Vanderbilt under Stassun. Although Farid was too advanced to qualify for a master’s degree through the bridge program, she participated in many of its activities. Looking back, Farid thinks her experiences at AAMU, UNH, and Vanderbilt show that a partnership between a predominantly white institution and a minorityserving institution is no panacea. “It defi-

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BLACK COLLEGES CAN’T DO IT ALL

At historically Black colleges and universities, tight funding threatens an extraordinary record of nurturing Black physicists

CREDITS: (PHOTO) MATTHEW ODOM; (ILLUSTRATION) C. SMITH/SCIENCE

By Jeffrey Mervis

Robert Dixon has spent more than 50 years mentoring physics students at historically Black colleges and universities.

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istorically Black colleges and universities (HBCUs) in the United States have had outsize success in launching Black students into physics. Although only 9% of all Black undergraduates attend the country’s 100 HBCUs, those schools for decades have awarded the majority of physics degrees earned by Black students. HBCUs also claim all the slots on a top 10 list of schools graduating the most Black physicists, despite having departments that are much smaller and have less funding than those at predominantly white institutions (PWIs). How do they do it? The key, say dozens of Black scientists who have worked at HBCUs or are knowledgeable about them, is that they provide a nurturing environment that addresses the academic, financial, emotional, and cultural needs of their students. But HBCUs’ ability to do so is threatened by declining overall enrollments over the past decade and the worsening of already serious financial constraints. In 2019, HBCUs garnered $341 million in federal research funding, down 15% from 2001; over the same 2 decades, the amount going to all U.S. universities grew by 65%, to $38 billion. The absolute number of Black students earning undergraduate physics degrees from HBCUs fell by half between 1996 and 2018, according to data from the American Institute of Physics (AIP). And HBCUs’ share of all Black physics graduates, which stood above 50% in 2006, was only 28% in 2019. (Only one-third of HBCUs offer an undergraduate degree in physics.) That shifting balance would matter less if the physics departments at the nation’s research heavyweights—all PWIs—were doing a better job of deploying their large research budgets and hefty endowments to fill the pipeline with Black physicists. But even those with the best records are falling far short of what’s needed to improve diversity. The data for the Massachusetts Institute of Technology (MIT), one of the nation’s leading universities, bear that out. Between 2012 and 2017, MIT awarded more undergraduate physics degrees to Black students— 12—than any other PWI, according to AIP statistics. Even so, none of the 42 physics majors graduating from MIT in 2017 was Black. By comparison, eight Black students earned physics degrees that year from Morehouse College, an all-male HBCU in Atlanta. That stark racial disparity is why Sylvester James Gates, an eminent theoretical physicist who is Black, views HBCUs as a precious resource for the community. “They are our intellectual lifeboats,” Gates said last year SCIENCE science.org

during an American Physical Society webinar on diversity that he chaired as APS president. “Investing in them is a bet on ourselves.” FOR HBCUs trying to build robust physics

programs, Morehouse has long been an exemplar. A 2020 report from AIP’s National Task Force to Elevate African American Representation in Undergraduate Physics & Astronomy (TEAM-UP) found the college produced 32 Black physics majors between 2012 and 2017, a dozen more than second place Alabama A&M University. A generation earlier, Morehouse had enjoyed similar success under Robert Dixon, a Black physicist who led its physics department from 1988 until 2004. “Bob Dixon has probably trained more African American physics undergraduates than anyone else in the country,” says Warren

“

Bob Dixon has probably trained more AFRICAN AMERICAN PHYSICS UNDERGRADUATES than anyone else in the country. WARREN BUCK, UNIVERSITY OF WASHINGTON, BOTHELL

Buck, a Black physicist and former chancellor of the University of Washington (UW), Bothell. “He’s underrated because he doesn’t look for glory. But he’s very effective,” adds Buck, a former chair of the physics department at Hampton University, an HBCU in Virginia. Now 80 and semiretired, Dixon has worked as a faculty member and administrator at a half-dozen HBCUs over more than 5 decades. Arguably his greatest success came at Morehouse, where he earned an undergraduate degree in 1964 and then returned 2 decades later to join its faculty. Its small physics program was limping along, he says, and after becoming chair he realized the only way to build it up was “to look for grant opportunities.” His success in winning federal funding allowed him to grow the number of faculty from three to 11, offer scholarships, and hire

staff to plan a range of events that raised the department’s campus profile. “We became a hub of activity,” he says, “and it drew students into the program.” Nicholas Fuller was one of them. Raised in Trinidad and Tobago by a single mother who regarded a good education “as the only path to success,” Fuller excelled at science in high school. When it was time to go to college, he chose Morehouse because it offered him a full scholarship. Dixon’s approach to training the next generation of physicists also resonated with him. “The level of nurturing is the key,” says Fuller, who went on to earn a doctoral degree in applied physics at Columbia University and now directs global hybrid cloud services for IBM. “If you failed an exam, Dr. Dixon let you know that you still had a bright future. Without that support, students lose confidence in their ability to become a scientist or engineer, especially if they don’t see many people in those jobs who look like them.” In 1996, Dixon won a $7.3 million grant from a U.S. Department of Defense (DOD) program designed to strengthen undergraduate science at HBCUs. “DOD’s original plan was to fund 20 schools,” he recalls. “But I asked for all of the money, on the grounds that we had the best proposal.” Dixon used the money to create the Center for Excellence in Science, Engineering, and Mathematics (CESEM) at Morehouse. CESEM provided full scholarships and intensive academic and career guidance to 50 Morehouse freshmen seeking an undergraduate degree in the natural sciences, math, and engineering, including 17 in physics. Some 85% had earned degrees by the end of the grant, and upward of 80% chose to continue to a graduate science, technology, engineering, or math (STEM) program. Two hundred additional Morehouse STEM majors were able to take advantage of a subset of those activities. The grant also supported a cohort of 50 ninth grade students from Atlanta public schools, providing academic and career counseling for the students, all of whom graduated, and professional development for their teachers. Although the program was not designed to be a recruiting device for Morehouse, some participants enrolled there and majored in science. Yet Morehouse’s success, like that of other physics departments at HBCUs, rested on a shaky foundation. In 2001, DOD officials declined to renew the 5-year grant that supported CESEM, and Dixon asked senior college administrators for internal funding to continue the program. But they turned him 4 MARCH 2022 • VOL 375 ISSUE 6584

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retired, said recently that seeing that initiative falter was one of his biggest regrets. Even at the time, Rockward and his colleagues say, Georgia Tech didn’t match the welcoming atmosphere of an HBCU. Rebuffed when they tried to join a study group, for example, the Black students converted a departmental storage room into a retreat they called “the Black Hole.” Moving to Morehouse required Rockward to make adjustments. “No startup package, and all I had to work with were undergraduates,” he recalls. But he won a string of federal grants that included collaborations with colleagues at researchintensive universities, which provided internships for students to apply what they were learning in class.

Waning clout

Physics B.A. degrees awarded to Black students

Historically Black colleges and universities (HBCUs) once accounted for more than half of all U.S. physics degrees awarded to Black undergraduates. That share has now declined to less than one-fifth of the total. 300 HBCU

Non-HBCU 262 214

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he recruited Willie Rockward, a Black physicist. Rockward had graduated from Grambling, where he was drawn into the field by the school’s small but nurturing physics department. “Dr. [Odom] looked like me,” Rockward says about the department’s longtime chair, Thomas Odom Jr., one of several Black faculty members in the department. (Black science faculty are often in the minority at HBCUs as well.) Rockward went on to earn his Ph.D. from the Georgia Institute of Technology (Georgia Tech), where he was part of a first ever cohort of five Black students recruited by physicist Henry Valk, a senior administrator. A few years later, Valk, who is white, recruited a second cohort of similar size. It was a short-lived attempt to diversify the physics department, and Valk, long since 962

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In 2011, Rockward became department chair, giving him a chance to revamp a sequence of three calculus-based courses with notoriously high attrition rates. He reshuffled the instructors to match their strengths with the content of each course. Their success in retaining students led to larger graduating classes in physics, a virtuous cycle that made it easier to attract more majors. Those and other moves helped Morehouse return to the top of the national rankings in producing Black physics majors. But eventually Rockward, like Dixon, felt that his efforts were not valued and that his career was stagnating. “I didn’t get promoted to full professor,” he says. “So I said, ‘OK, that’s your call.’ And I started to look around.” In 2019, Rockward landed at Morgan State University, an HBCU in Baltimore.

One big attraction was the chance to replicate his success at Morehouse on a larger scale. “[Morgan State] had a strong record in the 1980s for graduating African Americans before things started to slide, and they were interested in reviving that tradition,” he says. Even better, Rockward says, school administrators had vowed that Morgan State would become a Tier 1 institution by 2030, a designation based on the amount of its external funding. Rockward hopes meeting that goal will catapult Morgan State into the ranks of Georgia Tech and other heavyweight physics programs at PWIs. WARREN BUCK, former physics chair at

Hampton, is another Dixon protégé who built a successful program at an HBCU by following his mentor’s template—and by adding his own wrinkles. Buck was an undergraduate at Morgan State in 1966 when he met Dixon, then a new faculty member in his first academic job. “Bob convinced me I could do physics,” Buck says. He earned his Ph.D. from the College of William and Mary and joined the Hampton faculty in 1984. He expanded its physics department and also created a doctoral physics program, one of only five at an HBCU. Those efforts profited from the university’s proximity to the Department of Energy’s newly opened Thomas Jefferson National Accelerator Facility (JLab), home of the Continuous Electron Beam Accelerator Facility. Buck also won federal funding for a research center of excellence in nuclear and high energy physics, using the money to recruit faculty as well as to fund students to work with the world-class physicists at JLab. “I wanted to show that we could play with the big guys,” he says. That approach appealed to Devin Walker, now an assistant professor at Dartmouth College. In 1994, Walker was a Black high school student in Memphis, Tennessee, with his sights set on MIT. “I had the grades, they had lots of resources, and I knew that smart kids went to MIT.” MIT accepted Walker—but didn’t offer him a scholarship. So he sought out Buck during an event at Hampton, where Walker’s siblings were enrolled. Walker’s initiative— and resume—convinced Buck to offer him a full scholarship. In 2006, Walker became the first U.S.-born Black student to earn a physics Ph.D. at Harvard University, working under prominent particle physicist Howard Georgi, whom he had met during a JLab summer internship. Despite Buck’s success at Hampton—in 2001, five Black students earned physics Ph.D.s, an extraordinary feat the department repeated in 2002—he says the science.org SCIENCE

CREDITS: (GRAPHIC) K. FRANKLIN/SCIENCE; (DATA) NATIONAL CENTER FOR EDUCATION STATISTICS/INTEGRATED POSTSECONDARY EDUCATION DATA SYSTEM/COMPLETIONS 1999–2020 (2020 DATA ARE PROVISIONAL)

down, he says, forcing him to shut the center and lay off its five-person administrative staff. Without the scholarships and paid internships, students drifted into other fields. Over the next 2 years, Morehouse’s annual production of Black physics majors plunged from six, a number that had sustained its top ranking, to zero. In 2004, after 16 years as department head, Dixon threw in the towel and took a job at Grambling State University, a Louisiana HBCU. “It was very disappointing that the college didn’t give us a chance to continue what we were doing,” he says. Physicist Walter Massey, Morehouse’s president at the time, declined to comment. However, Dixon had sown the seeds for the program to thrive again when, in 1997,

At Morgan State University, a historically black university, physics professor Ramesh Budhani (left) works with undergraduate Don-Terry Veal Jr. in a spintronics lab.

PHOTO: MORGAN STATE UNIVERSITY

school’s senior administrators balked at supporting his vision for a more robust program. “Physics is expensive, and they didn’t see its value,” Buck says. Hampton officials declined to comment. But the online biography of its longtime president, William Harvey, emphasizes his concern for the bottom line by noting that “Dr. Harvey is an astute businessman who runs Hampton as a business for educational purposes.” Buck, who says he was “worn out and very frustrated” at Hampton, saw UW Bothell, an emerging 4-year college that caters to students from groups traditionally marginalized in science, as a chance to start over. Within a few years after he left to become UW Bothell’s first chancellor in 1999, the flow of Black graduate students into physics at Hampton had dried up. TOP U.S. RESEARCH universities have long

relied on HBCUs to be the first rung on the academic ladder for Black physicists. The success enjoyed by Dixon, Rockward, and Buck shows how crucial federal research grants are to that role. But those dollars are in short supply at HBCUs. In 2019, for exSCIENCE science.org

ample, North Carolina A&T State University led all HBCUs in winning federal research support, with $22 million. In contrast, five PWIs received more than $750 million each in federal research that year. In competing for those funds, HBCU faculty are at a decided disadvantage compared with their peers at PWIs. Heavy teaching loads often leave them little time to do the preliminary work needed to win a federal grant, and few HBCUs have the institutional funds to supplement any grants to support student research. In addition, the federal government prefers to back one-off experiments in education. “We don’t make long-term, sustained investments in STEM education like we do for research projects,” says physicist Claudia Rankins, former dean of science at Hampton who recently retired from the National Science Foundation after 2 decades of managing programs to broaden participation. “If you study some small particle, you can … be funded for decades as long as you show progress. But if you are proposing to do something in STEM education, or institutional capacity building, you’re fortunate to get 5 years of fund-

ing. And then you’re expected to move onto something else.” The TEAM-UP report calls for U.S. physics departments to double the number of Black majors by 2030. Reaching that goal will require PWIs to boost their output, and collaborations with HBCUs are one obvious route to success. But Marta McNeese, chair of the physics department at Spelman College, an all-women HBCU in Atlanta, says those relationships will need to be genuine partnerships, not a check-the-box exercise. “I’ve had people ask me to sign a letter of support [on a grant application], giving me 48 hours to sign, and telling me that all I need to do is send our students to their summer program,” McNeese says. “They want to address diversity, equity, and inclusion, but they don’t involve us in the planning.” The PWIs will also need to emulate the nurturing environment at HBCUs. “Our students are already stressed academically, politically, and economically,” McNeese says. “The role of an HBCU today is to give students a place where they can be themselves.” j 4 MARCH 2022 • VOL 375 ISSUE 6584

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CALLED TO

TEACH

Black physics Ph.D.s are more than twice as likely as other groups to teach in high schools and community colleges. For many, it’s a mission

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or years, Maritza Tavarez-Brown couldn’t talk about the end of her astronomy career without tears. She’d wanted to be an astronomer since high school. But she struggled in her introductory physics classes at Yale University. At one point, she remembers, the department chair told her she should reconsider her major. Determined, she transferred to New York City’s Hunter College, earned bachelor’s and master’s degrees in physics, and completed a Ph.D. on the properties of dark matter halos at the University of Michigan, Ann Arbor. She was offered a postdoc at the University of California, Berkeley, the kind of prestigious apprenticeship that could catapult her to an academic faculty position. She turned it down. She and her husband had just relocated with their 5-yearold daughter to Seattle, and they decided moving to the San Francisco Bay Area wouldn’t work for their family. That was more than 16 years ago, but Tavarez-Brown remembers it as if it were yesterday. “I was devastated,” recalls TavarezBrown, who is of Afro-Cuban descent. “In Spanish, we have this saying, ‘You’re work964

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ing so hard that you’re burning your eyelashes,’” she says. “I had done all this hard work, and now the thing that I was looking for … I can’t really do.” Instead, she took a position as a long-term substitute physics teacher at Forest Ridge School of the Sacred Heart, a middle and high school for girls in nearby Bellevue, Washington. Today, “Dr. T,” as her students call her, is still at Forest Ridge, and she is part of a long but little-known tradition in U.S. science education: For decades, Black Ph.D. scientists have opted to teach outside the ivory tower in proportions higher than any other race or ethnicity. They are more than twice as likely as their non-Black peers to teach at 2-year colleges, high schools, or other precollege institutions, according to an analysis of the National Science Foundation’s (NSF’s) Survey of Doctorate Recipients. The disparity is present across a range of disciplines, but it has been especially pronounced in the physical sciences, where roughly one in 10 Black Ph.D. holders teaches outside of 4-year higher learning institutions—and where in some years Black Ph.D. holders have been more than three times as likely as the average to hold those jobs. These trends

date back at least to 1999, the first year NSF published the relevant data. Education experts puzzle over the causes of the disparity. But for their part, TavarezBrown and other Black Ph.D. physicists who have made the leap say they are finding fulfillment on a career path that many science graduates never consider traveling. For the students they teach, their career choices mean a chance to learn from trained scientists—and to see new role models in science. THE RELATIVE SURPLUS of Black Ph.D. sci-

entists teaching in high schools and 2-year colleges has slipped under the radar of many researchers. Ebony McGee, an associate professor of diversity and science, technology, engineering, and math education at Vanderbilt University, calls it “extremely surprising.” But she sees a possible explanation, pointing to evidence that many Black scientists and engineers flee the ivory tower because they find the environment constraining, marginalizing, and systemically racist. “What it sounds like to me is that the structures don’t allow them to see themselves as authentically in [uniscience.org SCIENCE

IMAGE: VIRTUALPHOTO/ISTOCK.COM AND ELI_ASENOVA/ISTOCK.COM, ADAPTED BY C. SMITH/SCIENCE

By Ashley Smart

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PHOTO: PETER TEMPLIN

Maritza Tavarez-Brown left behind a budding career in astronomy and found fulfillment as a teacher.

versity] faculty positions as they do in community college or high school positions.” A close look at the NSF data suggests additional factors may be at work. The data indicate Black scientists who obtain doctorates are no less likely to teach at 4-year higher learning institutions than Ph.D. scientists from other racial and ethnic groups. (The NSF data don’t show what fraction of those faculty are at researchintensive as opposed to teaching-intensive schools.) Rather, for reasons that remain unclear, the surfeit of Black doctorates opting to teach at high schools and 2-year colleges appears to be offset primarily by a deficit of Black doctorate holders working in private industry. Kimberly Griffin, a professor of higher education, student affairs, and international education policy at the University of Maryland, College Park, cautions that it would be a mistake to assume that Black Ph.D. holders who opt for precollege and community college teaching careers are somehow settling. “This might be a very intentional choice,” Griffin says—“different than what they anticipated when they started [their Ph.D.], but still very intentional.” SCIENCE science.org

decision to pursue a teaching career was very much intentional. She realized toward the end of her Ph.D. that what she enjoyed more than crunching numbers and writing papers was talking to people. Both of her parents taught at community colleges, and she had an aunt and a cousin who taught at public schools. Plus, she had enjoyed the teaching she’d done as a graduate student—and she’d excelled at it, winning student teaching awards. It wasn’t that she lacked the research credentials to pursue a university faculty position. Her dissertation was on the dynamics of merging black holes, an area that would soon become one of the hottest in physics, with the dramatic first detection of gravitational waves from such mergers. In the run-up to academic hiring season, her adviser had even helped arrange for her to give a series of symposia and public talks—which, she says, were “very well received.” But Cohen Gibbons, who is Black, figured a high school teaching position would offer her a chance “to be much more than just somebody who’s delivering content”— to engage with students in areas such as social and emotional learning and social VANESSA COHEN GIBBONS’S

justice. So, just months before the academic hiring season began, she decided to apply exclusively for high school teaching jobs. Before long she had multiple offers. She accepted a position at the Garrison Forest School, a private, all-girls K-12 school in suburban Baltimore. (The high school teachers interviewed for this story all opted for private schools, noting, among other factors, that those jobs allowed them to bypass the time-consuming certification process many public schools require.) Cohen Gibbons’s realization, years into her graduate studies, that an academic research career wasn’t for her is hardly unusual. Studies have repeatedly found that, in aggregate, students tend to lose interest in faculty positions over the course of their graduate careers. In a 2017 study that surveyed more than 850 U.S.-based Ph.D. students in physics, chemistry, engineering, computer science, and the life sciences, nearly one-third of students who entered their Ph.D. programs expressing an interest in academic research careers had cooled on the idea by the time they neared graduation. The drop-off appears to be especially steep for women from underrepresented racial and ethnic groups. In focus group studies, these women are also more likely to credit “externally focused” values—such as a desire to mentor students or to solve health problems facing their communities—as motivating factors in their career choice. A study conducted by the American Institute of Physics similarly found that Black physics students were more likely than physics students of other ethnic groups to express a commitment to benefitting their community. Griffin, who co-authored some of those studies, thinks those values—particularly a desire to give back through education— could help explain the disproportionate numbers of Black Ph.D. scientists opting for careers in K-12 and community college teaching. “I could see someone getting a [science] Ph.D. and using that as their pathway to fulfill this cultural commitment.” Fana Mulu-Moore likely falls into that category. As a postdoctoral fellow studying solar physics at NASA’s Marshall Space Flight Center, she did a lot of outreach, speaking to and working with students at historically Black colleges and universities (HBCUs) in the southeast. Those interactions felt meaningful, she says. Meanwhile, she’d begun to feel burnt out by research, and she found it isolating being one of just a few Black women in her field. So in 2013, when Mulu-Moore took a year off to care 4 MARCH 2022 • VOL 375 ISSUE 6584

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for her newborn child, she decided she would pursue teaching jobs. After a summer stint as a high school physics teacher, she landed at Aims Community College, a small school in Greeley, Colorado, about 1 hour’s drive north of Denver. In the spring of 2021, she was promoted from an adjunct position to become the school’s only full-time physics professor. Mulu-Moore describes Aims as a small, nurturing community that reminds her of her own alma mater, Alabama A&M University, the HBCU where she earned both her bachelor’s degree and her Ph.D. Its student body, like those of many community colleges, is diverse—roughly 40% students of color, many of them first generation students. “It’s a great place to make an impact,” she says.

mathematicians into secondary school teaching. Some high school administrators the committee interviewed speculated that Ph.D. scientists would be “overspecialized and overprepared for teaching secondary school students.” They also noted they could not match the pay Ph.D.s could earn outside the classroom. Still, the council concluded that if high schools could woo Ph.D. scientists into their classrooms—and provide them with the necessary pedagogical training—the rewards would be plentiful. For Mark Hannum, a white physics teacher who serves as K-12 programs manager at the American Association of Physics Teachers and heads the science department at Thomas Jefferson High School for Science and Technology, an important advantage of having

From the lab to the classroom For decades, Black Ph.D.s in the physical sciences have opted to teach outside the ivory tower—largely at high schools and community colleges—in proportions higher than any other race or ethnicity. 16

Physical sciences Ph.D.s teaching at non−4-year institutions (%)

Asian

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8

AS FOR TAVAREZ-BROWN, she still feels

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TEACHERS WITH MULU-MOORE’S credentials

are a boon to community colleges and high schools. According to a 2005 report from the American Association of Community Colleges, only about one-quarter of the science faculty at 2-year colleges hold doctorate degrees. In high schools, most physics, chemistry, and earth science teachers lack even an undergraduate degree in the discipline they teach, let alone a doctorate. According to recent data from the American Institute of Physics, just 27% of high school physics teachers hold a degree in physics, and about 4% have doctorates. Two decades ago, the paucity of teachers trained in the sciences so troubled U.S. educators that the National Research Council convened a committee to explore the feasibility of recruiting more Ph.D. scientists and 966

scientific process over content memorization, he says, and they bring a keen understanding of what it means to produce knowledge through experimentation. The prospect of bringing research knowhow into the classroom appealed to Angela Meyer. After finishing a Ph.D. in astronomy at Georgia State University, Meyer—a biracial Black woman—took a tenure-track faculty position at Florida Gulf Coast University, where she expected to help develop a new earth and space sciences major and launch a new observatory. But those plans never panned out. After a conversation with a cousin-in-law who taught biology at a private high school, Meyer figured a private school setting might offer her the opportunities she sought. Meyer wound up at Culver Academies, a private boarding high school in northern Indiana. The job allowed her to pursue science without the constant pressure to write grants and publish. She developed a new astronomy curriculum, and she explored the use of classroom observatories—small, remote telescopes designed for educational use that can be controlled through the internet. One such observatory she’s worked with, a network of telescopes maintained by the HarvardSmithsonian Center for Astrophysics, can be used for classroom projects on finding and characterizing exoplanets. “It’s not research at the level I was doing,” Meyer says. But it allows her to combine her passion for working with students with her passion for working with data. “Those are my two great loves.”

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trained researchers in the classroom is that, unlike many other science teachers, they think of themselves as scientists. Hannum, who has bachelor’s and master’s degrees in physics, counts himself among that group. Having a teacher who identifies as a scientist can profoundly impact the way the students see science, Hannum says. It gives them a role model—and all the better, he says, if that role model is someone who can broaden students’ perceptions of who does science. To that end, he says, “having Black Ph.D.s working in schools is immensely valuable.” Having a scientific background can also affect how the teachers approach their work, Hannum says. “I think that mindset has subtle ramifications that spill over in a lot of ways.” For example, trained scientists are typically better prepared to emphasize the

twinges of regret. Once in a while, she’ll pull out her doctoral thesis and thumb through the pages, reminding herself of the work that went into it: the weeks in New Mexico learning to analyze data from the Very Large Array; the weeks in the Netherlands learning to reduce data from the Westerbork Synthesis Radio Telescope. For her, an academic research career was always the prize. It stings to know that the goal has likely drifted permanently out of her reach. But, like so many other Ph.D. physicists who have followed similar paths, she is ultimately at peace with the way things worked out. “To have a girl come up to you and be like, ‘I never thought I was good in science,’ or ‘I never thought I can do this and like it,’ … honestly, you’re not gonna get that being an astronomer,” she says. “I know I’m making an impact—right here, right now, for the future generation.” j Ashley Smart is associate director of the Knight Science Journalism Program at the Massachusetts Institute of Technology and a senior editor at Undark magazine. science.org SCIENCE

CREDITS: (GRAPHIC) K. FRANKLIN/SCIENCE; (DATA) NSF/SURVEYS OF DOCTORATE RECIPIENTS 1999, 2001, 2003, 2006, 2008, 2010, 2013, 2015, 2017, 2019

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MICHIGAN’S SURPRISING PATH TO

DIVERSITY Black graduate students find a nurturing culture in its applied physics program By Jeffrey Mervis

ILLUSTRATION: C. SMITH/SCIENCE

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oy Clarke missed the freewheeling atmosphere of the legendary Bell Telephone Laboratories after he left Bell to join the physics faculty at the University of Michigan (UM), Ann Arbor, in 1979. He realized that UM’s graduate physics program, as is the case at most universities, operated within tight disciplinary silos that prescribed what students should learn and whom they studied with. “It limited the scope of the research you could do, and its impact,” he says about an educational philosophy that he compares to the masterapprentice model of a medieval guild. Rather than trying to change age-old practices, Clarke won approval for a new graduate program in applied physics (AP). And in making graduate physics training far more collaborative and interdisciplinary, Clarke unwittingly also found a formula to improve its racial, ethnic, and gender diversity. “We found that our approach attracted a lot of interest from students of color,” he says. That’s a typical understatement by the white, Australian-born Clarke. Within a few decades of its 1987 debut, UM’s AP program was producing roughly 10% of all Black students earning U.S. physics Ph.D.s each year, according to a 2017 study by Julie Posselt, an education researcher at the University of Southern California, who is white, and colleagues at UM. Black, Latino, and Native American students made up as much as onethird of the program’s typical entering cohort compared with 5% nationally. And women SCIENCE science.org

comprised one-third of a typical graduating class, twice the national average. One factor behind those numbers, says Clarke, who stepped down as director in 2002 but maintains ties to the program, is that “we don’t engage in the hand-to-hand combat” so common among graduate programs. Instead of winnowing out any student who is struggling, he says, “we make it clear that we expect people who come here to succeed.” What Clarke and his successors have done, the study notes, is discard the traditional playbook for graduate training in physics “that had implicitly created barriers to access and inclusion for underrepresented students.” The new approach removes those barriers, Posselt explains, by “reconceptualizing the vision of the ideal student, empowering administrative staff to serve as cultural translators across racial and faculty-student boundaries, and creating a familylike climate.” Meeting the needs of students needs to be a priority, she adds, not an afterthought. SCIENCE SPOKE with eight alumni of UM’s AP

program to learn how those principles were implemented. One is Kim Lewis, now a professor of physics and associate research dean at Howard University, a historically Black institution in Washington, D.C. In 1997, Lewis was a physics major at Dillard University, a historically black college and university (HBCU) in her hometown of New Orleans, when she first heard about UM’s program. It checked most of her boxes for graduate school.

“I really still wanted to be an engineer,” Lewis recalls, “so an applied physics program was very appealing. I also knew that I wanted to be an academic, and nobody would question my qualifications if I had graduated from Michigan.” But Lewis also wanted to go someplace where she felt comfortable—and Ann Arbor wasn’t at the top of her list. “It would be the first time I had been in a school setting where the people did not look like me,” says Lewis, who attended an all-Black high school. “It also meant being away from home.” It helped that four of the eight students in her class were Black, and that many had attended HBCUs. And Lewis says she was grateful to Clarke for setting up meetings that might lead to a lasting relationship. “‘You’re about to be best friends,’” Clarke told Lewis when he introduced her to Adrienne Stiff-Roberts, who entered the program a year later. “And he was right.” After graduating with their Ph.D.s in 2004, both women have more than achieved the AP program’s goal of preparing its students for success. Lewis spent 11 years on the faculty at the Rensselaer Polytechnic Institute before moving to Howard in 2018. Stiff-Roberts immediately joined the faculty at Duke University, where she is now a professor of computer and electrical engineering. In addition to supporting students traditionally underrepresented in physics, Lewis says, the UM program has shattered the myth that a graduate physics program must sacrifice quality to achieve diversity. And she gives 4 MARCH 2022 • VOL 375 ISSUE 6584

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Clarke a huge amount of credit for that. “He’s a scholar, one of the best condensed matter physicists in the country,” says Lewis, who works in the same field. “He has great compassion for his students, but he’s never watered down the program.” THE UNIVERSITY’S sterling reputation also at-

tracted Mitaire Ojaruega. He entered the AP program in 2003 and soon benefited from another tenet of Clarke’s philosophy: ensuring staff and faculty work together to provide students with the help they need. The 11th of 17 children, Ojaruega was born in the United States and spent much of his childhood in Nigeria. But he attended high school in Washington, D.C., and enrolled at the University of the District of Columbia (UDC). “It was a very safe environment, and the professors were very supportive, but it gives you a narrow view of the world,” he says about UDC, an HBCU that focuses on undergraduate teaching and has a tiny research budget. Summer internships at Northwestern University and UM “were my first exposure to the big leagues,” Ojaruega recalls, and he chose UM over other top-tier graduate physics programs after a professor promised “to make sure you graduate.” Ojaruega benefited from that supportive environment, which he calls his “circle of trust,” after he initially failed his qualifying exams. That circle included Brad Orr, a white physicist who succeeded Clarke as director of the program. “Brad looked at my [undergraduate] transcript, saw that I hadn’t taken solid state physics, and suggested I sit in on those [undergraduate] classes I had missed,” Ojaruega recounts. Some students might have been too embarrassed to take a step back, he says, but he wasn’t. “And it helped a lot.” “Michigan was hard as hell,” Ojaruega adds. “They didn’t babysit you. But they also were invested in your success.” Another key member of his circle was Charles Sutton, who served as a recruiter for the program from 2001 to 2015. Sutton, who is Black, didn’t fit the stereotype of a departmental program staffer: “I had gold chains, an earring, and the whole bit,” he says. He was also an unlikely mentor: He was younger than many of the graduate students, having just earned a communications degree from UM, and he held down a weekend gig as a DJ. But Clarke regarded Sutton’s outgoing personality, along with his experiences growing up in Mississippi and as a student at an overwhelmingly white university, as valuable assets. When Clarke hired Sutton, “he told me to just be myself, and not change,” Sutton says. For the next 14 years, 968

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Sutton became the “eyes and ears of the program,” as one UM professor told Posselt, serving as an intermediary to help students and faculty better understand one another. Ojaruega had met Sutton at a recruitment fair and the two became good friends. “Sometimes I would pick him up at the library at 3 a.m.,” Sutton recalls. And after Ojaruega failed his qualifying exams, Sutton spoke up for his friend. “I had heard that some faculty members wanted to let him go,” Sutton recalls, “so I went to professor Orr and told him, ‘I know how hard [Ojaruega] is working. And I can promise you he won’t let you down.’” Sutton was right. Ojaruega passed his exams on the next try and sailed through the rest of his doctoral program, earning his degree in 2010. He has spent the past decade with the federal government and is now developing quantum sensor technology for the National Geospatial-Intelligence Agency within the Department of Defense.

“

Michigan was hard as hell. THEY DIDN’T BABYSIT YOU. But they also were invested in your success. MITAIRE OJARUEGA, DEPARTMENT OF DEFENSE

THE AP PROGRAM is an interdepartmental

program, not a free-standing department with its own assigned faculty. That arrangement is a boon to the type of interdisciplinary projects that Clarke encourages, with students free to choose an adviser from across the university. But it can backfire if those faculty members don’t share the AP program’s passion for putting students’ welfare first. That’s what happened to Bryan Ramson, who earned his undergraduate and master’s degrees from Howard. “I enjoyed being in the midst of high-achieving Black folks” and a predominantly Black faculty “committed to my success,” says Ramson, who is Black. That support evaporated, however, after he entered the AP program in 2011. Instead, Ramson says he went through “major culture shock” triggered by what he calls a “horrible”

environment in the university’s traditional physics department. “Some of the professors there are really old-guard, and very resistant to teaching” in ways that recognize different learning styles, Ramson says. “My professors were not interested in my success; their focus was on the research.” Sutton was there to reassure him that better times lay ahead. “It was the AP program staff, and Chuck in particular, who convinced me to stay whenever I would think about leaving,” Ramson says. He did, earning his Ph.D. in 2017. He’s doing a postdoc at Fermi National Accelerator Laboratory (Fermilab), where he began working as a graduate student in 2013. Kelly Nash, a professor at the University of Texas, San Antonio (UTSA), battled that same “old-guard” culture—and lost. “It was traumatic for me from the very beginning,” says Nash, a Black physicist who entered the AP program in 2000 after graduating from Dillard. The Ann Arbor campus was in turmoil as a lawsuit questioning the university’s use of race as a factor in admissions wended its way to the U.S. Supreme Court. In addition to being bombarded by the divisive rhetoric, Nash was also subject to pervasive racist stereotyping. “I remember taking a quantum mechanics class with three or four other Black students,” Nash recalls, “and our professor pulled us aside one day early in the semester. ‘I just wanted you to know that you’re all going to struggle in this class because you come from HBCUs,’ he said. He was basically saying that we didn’t belong. My science identity was constantly under assault.” Failing her qualifying exams turned out to be the final straw for Nash. “There was a huge debate about what should happen to me,” she says. “I had never failed a course [at UM] and never been on academic probation. But I had to fight to [even] get my master’s degree.” She departed feeling “exhausted and demoralized.” One year later she took another shot at graduate school and ended up at UTSA, which was starting a doctoral physics program. The fact that Latino students make up a majority of its undergraduate enrollment contributed to a welcoming environment that allowed her to thrive, she says. “Being a new program, they may not have had preconceived notions about African American students,” she adds. And her productivity was undeniable. “By the time I graduated [in 2009], I had 13 papers,” she says. A few years later she returned as a faculty member, earning tenure in 2016 and becoming a full professor in 2020. science.org SCIENCE

PHOTO: (OPPOSITE PAGE) ERIC BRONSON/UNIVERSITY OF MICHIGAN

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By putting students first, University of Michigan, Ann Arbor, physicist Roy Clarke achieved unprecedented diversity in the applied physics graduate program he created.

Undeterred—or perhaps driven—by their experiences at UM, both Ramson and Nash have spent years working to increase diversity in physics. Ramson co-directs Fermilab’s long-running Saturday morning physics program for Chicago-area students and is a member of Change-Now, a collective of young Black physicists who are pushing Fermilab, the Department of Energy’s leading high energy physics facility, to improve equity and social justice within the profession and in the community. Nash is involved in several programs to attract more women and people of color into science. For example, she has helped her university participate in a national program sponsored by the American Physical Society that finds spots for qualified students from underrepresented groups who have been passed over by other graduate physics programs. UM’S AP PROGRAM has continued to evolve.

Cagliyan Kurdak, a Turkish-born physicist who joined the department in 1998 and became the program’s director in 2010, added a component for students who need to strengthen their academic background before diving into a doctoral program. Students from groups underrepresented in science receive 2 years of funding while they earn a master’s degree, and over the years two-thirds have transitioned into a doctoral program, where their 80% completion rate is more than 20 percentage points higher than the national average. But Kurdak and his team can’t rest on their laurels. “Creating conditions to support access and inclusion is not a one-time event, but an ongoing struggle,” Posselt wrote in her 2017 study. Posselt flagged two recurring challenges. One is what she calls the “negative racial climate” for many students of color at UM, a predominantly white institution. Those whom Posselt interviewed also spoke about a “negative feedback loop, in which poor representation of women and students of color raises red flags for prospective students … and deters them from matriculating.” Posselt says the UM AP program has replaced those red flags with a welcome sign through its “commitment to diversity.” Its interdisciplinary approach to science, flexible admissions criteria, and family atmosphere are baked into the AP program, agrees Sutton, who went to the White House as Clarke’s guest when, in 2010, Clarke received the Presidential Award for Excellence in Science, Mathematics, and Engineering Mentoring. “They just care so much about people,” says Sutton, now a recruiter for Thermo Fisher Scientific in Atlanta, about his former colleagues. “And in the end, that is what has made them so successful. I hope the program lasts forever.” j SCIENCE science.org

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INSIGHTS

PERSPECTIVES ANIMAL BEHAVIOR

Anthropogenic influences on bee foraging By Dave Goulson and Elizabeth Nicholls

B

ees are highly specialized insects, feeding almost exclusively on nectar and pollen from flowers, and in so doing contributing substantially to the pollination of wild plants and crops. Flying to and from patches of flowers is energetically costly, particularly for relatively large bees such as honey bees and bumble bees that live on a knife-edge in terms of balancing their energy intake. 970

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Suboptimal foraging conditions can result in a net loss of energy over the course of a foraging trip. Bees have evolved numerous adaptations to efficiently locate and extract floral resources, including impressive learning, navigational, and communication abilities. These are key to their survival and successful reproduction, because the number of offspring that can be provisioned is determined by the food surplus adult bees can gather. However, these foraging adaptations can be affected by anthropogenic

factors, but there are opportunities to minimize negative effects. The arrival of the Anthropocene has brought with it considerable challenges for wild bees. In particular, the spread of industrial agriculture has profoundly altered the landscape across large portions of the globe, often reducing the availability and diversity of floral resources. The most widely grown School of Life Sciences, University of Sussex, Brighton, UK. Email: [email protected] science.org SCIENCE

PHOTO: CYRIL RUOSO/MINDEN PICTURES

Efficient foraging is vital to bee fitness but is challenging in the Anthropocene

GRAPHIC: N. CARY/SCIENCE

A honey bee (Apis mellifera) collects pollen from a common sunflower (Helianthus annuus) and must then navigate back to its hive and communicate with others about this food source.

crops (cereals such as wheat, rice, and corn, comprising 79% of global crop area) are wind-pollinated, and hence provide minimal resources for pollinators. When grown in large monocultures, with extensive herbicide use to eliminate most weeds, the farmed landscape can be almost devoid of flowers. Although some crops such as canola, sunflowers, and many fruits and vegetables do require pollination and offer floral resources for bees, this situation also poses challenges. A field of canola, for example, can provide a glut of food for pollinators for a short period of perhaps 3 weeks, but very little before or afterward. A similar situation prevails in fruit orchards following the short blooming period. Honey bees may benefit from such highly clumped resources more than other bee species because they can recruit nestmates to this bounty by their waggle dance, have relatively long foraging ranges, and are able to store excess resources over the long term in their hive in the form of honey and beebread. However, it has been repeatedly demonstrated that pollinator diversity is key to efficient pollination service delivery, and that relying on a single domesticated bee species can limit crop yields (1). A further challenge for bees foraging in farmland is exposure to agrochemicals. Samples of honey commonly contain 10 or more pesticides in complex combinations, often including potent insecticides such as neonicotinoids (2). Bee exposure can occur in many ways, not just through feeding on treated crops, including contamination of wildflowers and woody plants in field margins and pollution of water sources. Neonicotinoids are neurotoxins, harming bees at concentrations commonly detected in the pollen and nectar of both treated crops and wildflowers (typical range 1 to 20 parts per billion). Sublethal effects include impaired navigation, which increases the frequency with which honey bees become lost when foraging (3). Neonicotinoids also impair learning of associations between scents and floral rewards, a vital skill that bees use to identify the most rewarding flowers. Exposure to neonicotinoids reduces the proportion of workers that bring back pollen to the nest, and the amount of pollen gathered per trip, perhaps by impairing motor skills needed in pollen collection (3). Other types of insecticide, such as sulfoxaflor, flupyradifurone, and even the herbicide glyphosate have also been found to have negative sublethal impacts on aspects of foraging behavior, memory, and learning SCIENCE science.org

in various bee species (4) (see the figure). These sublethal effects of pesticides on behavior are rarely evaluated by regulatory tests, which typically focus on short-term effects on bee mortality, yet sublethal effects on foraging efficiency could profoundly reduce colony and nesting success. For example, if foragers regularly become lost when foraging, a social bee nest may quickly weaken and die as worker numbers fall. For solitary bees, which must single-handedly collect all food resources to provision their individual nests, the impact of inefficient foraging on reproduction and population size is likely to be even more pronounced (3). Furthermore, pesticides are applied in formulations con-

food. For some solitary species with foraging ranges of less than 100 m, new developments such as buildings or roads can represent considerable barriers to foraging. Pollutants and the disturbance associated with industry and transport are likely to have an impact on bees, in terms of the availability of both food and suitable nesting sites. Bees have been found to avoid foraging in areas affected by turbulence from passing traffic (7), and diesel exhaust emissions degrade floral odors, rendering it harder for bees to use sensory cues to detect and recognize rewarding flowers (8). Contamination of pollen and nectar with particulate pollution can expose bees

Anthropogenic factors that affect bee foraging efficiency Anthropogenic changes can affect the ability of bees to efficiently forage for food, which can reduce the survival of solitary and social bees.

! Habitat fragmentation Agriculture and/or urbanization cause bees to travel further from their nest to find food. This could especially affect smaller species or those exposed to pesticides and/or disease.

Exposure to pesticides and other chemicals Pesticides can have sublethal effects on bee behavior that impair navigation, memory, and learning. So-called "inert" ingredients can also affect bee foraging.

taining “inert” ingredients that are also not subject to regulatory tests. Recent studies reveal that surfactants used in “Roundup” (glyphosate-based herbicide) and “Amistar” (widely used fungicide based on azoxystrobin) are toxic to bumble bees (5). For example, the alcohol ethoxylate surfactants in Amistar cause gut damage, reducing appetite and foraging, and ultimately leading to 30% mortality in bumble bees (5). Farming is not the only anthropogenic land use that has consequences for bees and their capacity to forage efficiently. Biodiverse, flower-rich natural and seminatural habitats may be destroyed or fragmented as a result of spreading urbanization, the building of factories, transport infrastructure, golf courses, and much more (6). Such habitat fragmentation means that bees often need to fly further to find

Exposure to pollutants Airborne pollutants affect the detection of floral odors and learning about floral rewards. They can also impair flight capacity and navigation.

Climate change Higher temperatures and rising CO2 affect floral traits, such as flower number, nectar production, and protein content of pollen, which influence bee foraging choices.

to a range of industrial pollutants, including metals such as manganese, copper, and lead, and these have been found to have various behavioral effects, including inducing foraging at an earlier age in honey bee workers and reducing the number of foraging trips made per bee (7). Bees visiting flowers also risk infection with diseases, many of which are transmitted by shared contact with flower surfaces. Although a naturally occurring phenomenon to which bees have some behavioral adaptations, the threat to bee health has been greatly exacerbated by the transport of domesticated bees around the globe, leading to the introduction of diseases to which native bees have limited defenses. These emerging diseases can have profound effects on foraging ability and can alter floral preferences, as well as increasing mortality 4 MARCH 2022 • VOL 375 ISSUE 6584

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such as the national ban on urban pesticide use in France, further enhance the value of urban areas for foraging bees. There is evidence to suggest that honey bee and bumble bee colonies may fare better in urban areas than in agricultural landscapes, thought to be due in part to the greater diversity and availability of floral resources throughout the year (6, 11). Increased public interest in bee declines has also led to a rise in urban beekeeping, with many businesses and hotels offering space for urban hives through a desire to help declining bee populations. However, increasing the number of managed hives does nothing to support wild bee populations, and actually raises concerns regarding competition with wild bees for floral resources and a potentially exacerbated risk of disease transmission at flowers. Finding ways to support wild pollinators in farmland is an even greater challenge. Agri-environment schemes to support specific management for pollinators may enhance bee populations at a local scale but have not halted overall patterns of decline (15). The design and success of such schemes could be improved through a better understanding of the dietary needs and foraging behavior of bee species other than just honey bees and bumble bees, which currently benefit most from interventions such as wildflower strips. However, systemic change with a move toward regenerative farming practices—including use of legume cover crops, higher crop diversity, and reduced or eliminated pesticide use—are likely to be necessary to sustain the thriving and diverse wild pollinator community needed to provide a resilient pollination service for both crops and wildflowers. j REF ERENCES AND NOTES

1. L. A. Garibaldi et al., Front. Ecol. Environ. 12, 439 (2014). 2. F. Sanchez-Bayo, K. Goka, PLOS ONE 9, 94482 (2014). 3. T. J. Wood, D. Goulson, Environ. Sci. Pollut. Res. Int. 24, 17285 (2017). 4. J. Hernández, A. J. Riveros, M. Amaya-Márquez, J. Insect Conserv. 25, 683 (2021). 5. E. A. Straw, M. J. F. Brown, Sci. Rep. 11, 21653 (2021). 6. K. C. R. Baldock, Curr. Opin. Insect Sci. 38, 63 (2020). 7. B. B. Phillips et al., J. Appl. Ecol. 58, 1017 (2021). 8. R. D. Girling, I. Lusebrink, E. Farthing, T. A. Newman, G. M. Poppy, Sci. Rep. 3, 2779 (2013). 9. H. Koch, M. J. F. Brown, P. C. Stevenson, Curr. Opin. Insect Sci. 21, 60 (2017). 10. C. C. Nicholson, P. A. Egan, Glob. Change Biol. 26, 380 (2019). 11. D. Goulson, Silent Earth (HarperCollins, 2021). 12. H. Siviter et al., Nature 596, 389 (2021). 13. P. J. Kolano, M. Røyset Aarønes, K. Borgå, A. Nielsen, J. Pollinat. Ecol. 28, 138 (2021). 14. L. Tong, J. C. Nieh, S. Tosi, Chemosphere 237, 124408 (2019). 15. C. Geppert et al., J. Appl. Ecol. 57, 1818 (2020).

NEUROSCIENCE

Addicted to dreaming How does dopamine, the brain’s pleasure signal, regulate the dream stage of sleep? By Elda Arrigoni1 and Patrick M. Fuller2

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ow human brains regulate sleep remains an enduring puzzle (1). How sleep subserves human dreaming— rapid eye movement (REM) sleep—is especially puzzling. There is considerable mechanistic understanding of the synaptic, cellular, and circuit bases of REM sleep (2, 3). However, despite pharmacological evidence that dopamine (DA) can potently modulate REM sleep, this neurotransmitter is conspicuously absent from most prevailing REM sleep circuit models. DA is historically associated with pleasure and addiction. On page 994 of this issue Hasegawa et al. (4) report that the release of DA in the basolateral amygdala (BLA), a brain structure associated with emotional processing, can trigger REM sleep in mice and also that selective manipulation of DA release within the BLA can trigger cataplexy, which occurs in the sleep disorder narcolepsy and manifests as a crippling pathologic intrusion of REM sleep into wakefulness that results in loss of postural motor control. Although a role for DA has long been suggested in the regulation of sleep, including REM sleep, its precise contribution (and source) has remained enigmatic. An important clue emerged from a recent study in

ACKNOWL EDGMENTS

E.N. is supported by a UK Research and Innovation Future Leaders Fellowship (MR/T021691/1).

10.1126/science.abn0185

Rapid eye movement (REM) sleep is a behavioral state that is conserved across the animal kingdom, yet the biological purpose it serves remains unknown. science.org SCIENCE

PHOTO: KURT_G/SHUTTERSTOCK

(9). For example, infection with the parasite Nosema ceranae, which originated in Asia and is now common throughout the world, reduces honey bee foragers’ homing ability. If all this were not enough, climate change is likely to add further to the challenge of efficient foraging in bees. Being large and furry, bumble bees are adapted to cool, temperate climates and can overheat in warm weather, becoming unable to forage. In specialist bees that have a narrow range of food plants, the timing of emergence of bees and flowering may become uncoupled, and their offspring may lack the ability to digest and develop on alternative pollen sources (10). Rising CO2 concentrations can reduce the protein content of pollen, while extreme climatic events such as heat waves, fires, and droughts are likely to alter the ability of plants to produce floral resources, and such effects will undoubtedly get worse in coming decades (10). There are also likely to be other anthropogenic factors that affect bee foraging that are not yet recognized or properly researched. For example, electromagnetic radiation (radio waves, microwaves, and fields around high-voltage electricity lines) may plausibly interfere with the ability of bees to detect and use Earth’s magnetic field for navigation, but robust experiments are lacking. Does particulate pollution block insect trachea? Are insects affected by other environmental pollutants, such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), or any of the approximately 144,000 different manufactured chemicals that enter the global environment (11)? Furthermore, very little is known about how these stressors interact (12). Are effects additive or synergistic? In bumble bees, for example, exposure to sublethal concentrations of the insecticide clothianidin has been shown to cause a temperature-dependent decrease in foraging duration (13). In honey bees, a recent study using flight mills has shown that nutritional stress combined with exposure to the pesticide flupyradifurone increased mortality and also increased flight velocity (14). These are just a few examples of the multitude of combined stressors bees are currently exposed to, and the pace of change is such that ecologists and ecotoxicologists cannot keep up with testing the impact of new formulations as they come to market. There are opportunities to mitigate some of the pressures on bees. There is growing interest in planting bee-friendly flowers in gardens and other urban greenspaces, and in managing road verges to encourage wildflowers (despite risks associated with foraging in high traffic areas). Initiatives to reduce or eliminate pesticide use in urban areas,

GRAPHIC: A. MASTIN/SCIENCE

mice showing that DA neurons the BLA itself. One possibility is A putative circuit model of REM sleep within the midbrain ventral that disinhibition of non–DRD2Dopamine neurons within the ventral tegmental area (DAVTA neurons) project to tegmental area (VTA), a brain expressing BLA neurons—which and inhibit locally projecting basolateral amygdala (BLA) neurons that express region long associated with rethe authors suggest may occur DA receptor D2 (DRD2). This disinhibits the BLA efferent pathways, potentially ward and addiction, had higher after DA-induced inhibition of including projections to the central amygdala nucleus (CeA). In this putative firing rates during REM sleep DRD2-expressing BLA neurons— circuit model, activation of CeA g-aminobutyric acid–specific (GABAergic) neurons could promote rapid eye movement (REM) sleep and cataplexy through and wakefulness than during could, in turn, activate inhibitory projections to pontine and medullary cell populations. non-REM (NREM) sleep and g-aminobutyric acid–specific also increased their activity be(GABAergic) neurons of the fore NREM-to-REM transitions central amygdala nucleus (CeA) Anterior Midbrain Posterior (5). Building on these important that normally serve to inhibit Amygdala observations, Hasegawa et al. key pontine cell groups involved Midbrain sought to understand if and how in REM motor control (9–11) Pons these “REM-on” dopaminergic (see the figure). It also remains VTA (DAVTA) neurons might conunclear why the amount of DA tribute to the regulation of REM released in the BLA by positive sleep. To do so, they used a G proemotions (e.g., when mice are VTA tein–coupled receptor (GPCR) fed chocolate) is greatly ampliVTA DA activation–based (GRAB) sensor fied in orexin-deficient mice. The for DA (GRABDA) to assess DA reauthors propose that this could lease at known postsynaptic tarbe the result of disinhibition of gets of DAVTA neurons, including DAVTA input to the BLA or could Amygdala Pons the BLA, in mice. result from plastic changes in The GRABDA tool permits the DA system—e.g., the number precise temporal-spatial assessof aminegic neurons is increased ? ments of DA concentrations in narcoleptic humans who are GABA DRD2 GABA within the brain (6), even during orexin deficient (12, 13). Pontine complex behaviors. The authors Another remaining mystery is REM-off ? could thus ascertain extracelluwhy drugs that increase extracelneurons CeA lar DA concentrations within the lular DA (e.g., modafinil) and are BLA BLA and other brain regions as used clinically to enhance wakeGlutamate mice cycled through the stages fulness in people with disorders Spinal motor neurons of wakefulness, NREM sleep, like idiopathic hypersomnia and and REM sleep. From these renarcolepsy do not have an effect cordings, they determined that DA was tranet al. demonstrated that DA release into the on cataplexy and REM sleep. Future studsiently increased in both the BLA and the BLA precedes cataplexy in narcoleptic mice ies should also examine the role of BLA DA nucleus accumbens (NAc) preceding NREM(which are deficient in orexin, a neuropeptide signaling in shaping fundamental features of to-REM transitions. Subsequent photostimuexpressed in cells of the lateral hypothalamus REM sleep, such as its homeostatic regulalation of DAVTA terminals in the BLA but not that regulates wakefulness and appetite). tion, which is a process that remains incomin the NAc produced a rapid transition to They also found that the surge of DA in the pletely understood. The study of Hasegawa REM sleep. The authors then acutely and seBLA—but not in other brain regions tested— et al. provides fresh insights into the control lectively inhibited DA receptor D2 (DRD2)– was greater in the narcoleptic mouse model of both REM sleep and cataplexy by DA, and expressing BLA neurons in vivo using an opthan in wild-type littermates. The authors their findings raise the intriguing possibility togenetic tool (7), which triggered, albeit with then stimulated DAVTA neurons in nonnarcothat DRD2 BLA neurons could be a selecsome delay, an NREM-to-REM transition and leptic mice, which express normal amounts tive druggable target for treating debilitating increased total REM sleep time. Together, of orexin, producing a transient increase in symptoms in a wide range of REM sleep disthese findings reveal a central role for DAVTA DA within the BLA and inducing cataplexy. orders, including cataplexy in narcolepsy and neurons, DA neurotransmission, and inhiGiven the normal concentrations of orexin in other disorders, such as Parkinson’s disease, bition of DRD2-expressing BLA neurons in these mice, this is a striking finding and one in which DA signaling is disrupted. j the regulation of REM sleep, including the that may require a reconsideration of the preREF ERENCES AND NOTES triggering of REM sleep onset. These results vailing neurobiological models of cataplexy in 1. D. Kennedy, C. Norman, Science 309, 75 (2005). therefore provide a cellular and circuit subthe context of narcolepsy because cataplexy is 2. E. Arrigoni, M. C. Chen, P. M. Fuller, J. Physiol. 594, 5391 (2016). strate for findings from previous studies not known to occur spontaneously in orexin3. J. Peever, P. M. Fuller, Curr. Biol. 27, R1237 (2017). showing that the pharmacological manipulacompetent animals, including humans. 4. E. Hasegawa et al., Science 375, 994 (2022). tion of brain DA (e.g., DA-depleted mice and Although the authors’ data firmly support 5. A. Eban-Rothschild, G. Rothschild, W. J. Giardino, J. R. Jones, L. de Lecea, Nat. Neurosci. 19, 1356 (2016). the use of DRD2 agonists) can substantially a role for DAVTA neurons in the control of 6. F. Sun et al., Cell 174, 481 (2018). alter REM sleep (8). REM sleep, how DRD2-expessing BLA neu7. O. A. Masseck et al., Neuron 81, 1263 (2014). 8. K. Dzirasa et al., J. Neurosci. 26, 10577 (2006). Can manipulation of DRD2 neurons in the rons are functionally related or connected 9. M. B. Snow et al., J. Neurosci. 37, 4007 (2017). BLA produce cataplexy in mice? Hasegawa with cell groups located in the pontine and 10. C. E. Mahoney, L. J. Agostinelli, J. N. K. Brooks, B. B. medullary brainstem, which were previously Lowell, T. E. Scammell, J. Neurosci. 37, 3995 (2017). 11. E. Hasegawa et al., Proc. Natl. Acad. Sci. U.S.A. 114, 1 Department of Neurology, Beth Israel Deaconess Medical established to be necessary for the generation E3526 (2017). Center and Harvard Medical School, Boston, MA, USA. of REM sleep and REM paralysis, remains 12. P. O. Valko et al., Ann. Neurol. 74, 794 (2013). 2 Department of Neurological Surgery, University of 13. J. John et al., Ann. Neurol. 74, 786 (2013). uncertain. Notably, BLA DRD2-expressing California, Davis School of Medicine, Davis, CA, USA. Email: [email protected]; [email protected] neurons do not appear to project beyond 10.1126/science.abo1987 SCIENCE science.org

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ferent identities. Thus, Omary et al. called them “transition stem cells”; they are the intermediary stage between phloem parenchyma cells and shoot-borne root primordia initial cells. This transitional state served as a platform to identify potential regulators, including a transcription factor belonging to the LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family (5) that the authors By Lidor Shaar-Moshe1,2 and between lateral root and shoot-borne root named SHOOTBORNE-ROOTLESS (SBRL). Siobhan M. Brady1,2 primordia would demonstrate conserved They demonstrated that SBRL is necessary mechanisms of root primordia. As the shootfor shoot-borne root, but not lateral root, inilants continuously elaborate their borne root primordia undergoes cell pattiation in tomato. The function of SBRL orform through the production of new terning, cells within the primordia respond thologs was conserved in other dicotyledonorgans, including different types of to auxin or cytokinin or both. In tomato latous species, potato, and A. thaliana and likely roots. A primary root emerges from eral root initiation, there is no overlap in the conserved in sweet potato, white bean, and a seed, and lateral roots develop postcytokinin- and auxin-responsive domains, the monocot sorghum. Monocots and dicots embryonically from the primary root similar to Arabidopsis thaliana (4). Thus, the had a most recent common ancestor ~150 axis. In addition, plants can generate a varicells and hormonal signaling that give rise to million years ago. The identical function of ety of shoot-borne roots. These are called adshoot-borne and lateral root primordia differ, this gene across such a long evolutionary ventitious roots (1) in dicotyledonous plants, suggesting that these two developmental protime demonstrates substantial conservation. such as tomato, or crown and brace roots grams are different. The analysis by Omary et al. of tomato (2) in monocotyledonous cereal crops, such Omary et al. carried out single-cell tranSBRL and its orthologs indicated that as maize or sorghum. But the developmenscriptome profiling of shoot-borne root prishoot-borne and lateral root developmental mechanisms associated with shoot-borne mordia over developmental time, which retal programs are quite different. However, root initiation as well as the conservation of vealed a group of cells with an ephemeral cell a phylogeny of LBD transcription factors this program with lateral and wound-induced identity. These cells share a transcriptional demonstrated that SBRL belongs to subclass roots are largely unknown. On page 993 of enrichment for root stem cell regulators, and IIIB of LBD transcription factors. When asthis issue, Omary et al. (3) report a conserved a developmental trajectory analysis identisembling this phylogeny, the authors noted developmental pathway for shoot-borne fied this cell group as a branch point for difthat a subclass IIIB gene was nearly always roots across flowering plants located next to a closely related and a conserved “superlocus” subclass IIIA gene, which they that regulates the initiation of called an LBD superlocus, with Formation of specialized root types specialized root types. conserved regulatory elements. The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) superlocus regulates root Lateral roots derive from Subclass IIIA genes are associdevelopment by inducing a transition state. Lateral root initiation depends on pericycle cells that flank xylem ated with lateral root initiation expression of LBD IIIA genes in the xylem pole–pericycle. Shoot-borne root initiation cells (which transport water) in maize and A. thaliana (6). depends on expression of LBD IIIB genes in phloem-associated cells. Both LBD IIIA and within the root. By contrast, Do subclass IIIA genes also IIIB genes are required for initiation of wound-induced roots. Omary et al. demonstrate that regulate a transition stage in in tomato (Solanum lycoperlateral root initiation? Omary Shoot-borne root sicum), shoot-borne roots deet al. found that expression of IIIA IIIB rive from cells located around a large proportion of a gene the phloem (which transports set that defines the transition nutrients), called the phloem state was induced in analogous parenchyma. Hormone (auxin stages of lateral root initiation. and cytokinin) signaling conMutation of the tomato subtributes to the formation of a class IIIA gene BROTHER OF properly patterned root priSBRL (BSBRL) and its paralog Xylem mordia, which includes the BSBRL2 reduced lateral root formation of a stem cell niche initiation, with no change to Pericycle and asymmetric divisions of shoot-borne roots. Mutation of Wound-induced root Cambium IIIA IIIB stem cells to produce root cell the A. thaliana subclass IIIA Endodermis or types. To determine how fields gene resulted in an equivalent starch sheath of hormone-responsive cells phenotype. The local duplicaPhloem change through shoot-borne tion that gives rise to these Lateral root IIIA IIIB root primordia development, neighboring subclass IIIA and Omary et al. used hormoneIIIB genes therefore allowed response markers. Similarity the evolution of two indepenin the cellular response fields dent root-initiation programs from distinct parts of the plant 1 Department of Plant Biology, University body across diverse flowering of California, Davis, Davis, CA, USA. plant species. 2 Genome Center, University of California, A third wound-induced root Davis, Davis, CA, USA. Email: lshaar@ ucdavis.edu; [email protected] type is critical to agriculture PLANT BIOLOGY

Forming roots from shoot

Uncovering the genes responsible for different types of roots will transform aspects of plant agriculture

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and horticulture, as well as to the application of gene editing. Gardeners are familiar with the process of taking cuttings from plants to propagate a new plant asexually. Cuttings are the basis of the horticulture industry and involve the induction of roots from cut pieces of a plant stem that will eventually regenerate a whole plant. This is a form of asexual reproduction and is important for the propagation of flowering plants for which sexual reproduction can take years to decades. Gene editing in most species to improve plant performance in shorter times than traditional plant breeding approaches (7) requires transformation through the induction of root tissue (a wound-induced root) from a mass of undifferentiated cells called a callus and subsequent root initiation, followed by shoot initiation. Omary et al. showed that subclass IIIA and IIIB genes are also required for wound-induced root initiation (see the figure). Mutation of the subclass IIIB SBRL in potato interfered with root induction from callus. In tomato, simultaneous mutation of SBRL and the class IIIA BSBRL and BSBRL2 genes resulted in an inability to form woundinduced, shoot-borne, and lateral roots. Roots are incredibly important to plant survival owing to their roles in the transport of water and mineral nutrients and the provision of mechanical support. The discovery of this superlocus provides a beautiful example of how localized gene duplications produce a more complex plant form. The origin of cells that undergo the transition to a specialized root as well as the patterning events associated with generating the primordia can be distinct, but the IIIA and IIIB LBD transcription factors execute the same transitory state to produce different root types. Determining whether differences in class IIIA and IIIB transcription factor expression or activity underlie differences in the capacity for plant transformation or the production of cuttings will further strengthen the impact of the findings of Omary et al. The production of different root types is an important component of a plant’s ability to successfully respond to stresses in their environment, including withstanding flooding and high wind. In the face of climate change, subclass IIIB transcription factors may become important target genes to manipulate and produce plants with an increased capacity to adapt to adverse weather conditions. j REFERENCES AND NOTES

1. B. Steffens, A. Rasmussen, Plant Physiol. 170, 603 (2016). 2. A. N. Hostetler et al., Curr. Opin. Plant Biol. 59, 101985 (2021). 3. M. Omary et al., Science 375, eabf4368 (2022). 4. B. Müller, J. Sheen, Nature 453, 1094 (2008). 5. A. S. Chanderbali et al., Mol. Biol. Evol. 32, 1996 (2015). 6. Y. Okushima et al., Plant Cell 19, 118 (2007). 7. R. A. Nasti, D. F. Voytas, Proc. Natl. Acad. Sci. U.S.A. 118, e2004846117 (2021). 10.1126/science.abo2170 SCIENCE science.org

ULTRACOLD CHEMISTRY

Toward a coherent ultracold chemistry Magnetic fields can be used to change chemical reaction rates by a factor of 100 By Simon L. Cornish1 and Jeremy M. Hutson2

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ollisions are fundamentally important to the study of atomic and molecular gases. For example, they dictate the lifetimes of ultracold samples and the efficiency of cooling by evaporation. Ultracold collisions are largely governed by quantum mechanics and are very sensitive to electromagnetic fields. Harnessing subtle interference effects, such as Feshbach resonances, has allowed the control of atomic collisions and revolutionized the study of atomic physics (1). Ramping an applied magnetic field across such a resonance is the key step in the formation of ultracold diatomic molecules (2). Opportunities for a broad range of scientific studies and applications will open up if molecular collisions can be controlled in the same way as atomic collisions. On page 1006 of this issue, Son et al. (3) used a magnetic Feshbach resonance to offer better-than-ever control over ultracold reactive collisions between sodium (Na) atoms and sodium-lithium (NaLi) molecules. Ultracold molecule-molecule collisions have turned out to cause unexpectedly fast losses of molecules from the optical traps used to confine them (4–6). This loss happens even in systems in which two-body chemical reactions between the colliding species are not allowed (7). Most experiments have observed loss rates close to the so-called universal rate, at which all colliding pairs that reach short range are lost. The short-range loss is usually explained in terms of the formation of long-lived “complexes” during collisions (8). These complexes can be destroyed in a variety of ways, including by chemical reaction or absorption of light from the trapping laser (9). However, there is conflicting evidence on the lifetimes of the complexes and the mechanisms of their loss (5, 6, 10, 11). Feshbach resonances occur at magnetic fields where a state of the collision complex has the same energy as the colliding 1

Department of Physics, Durham University, South Road, Durham DH1 3LE, UK. 2Department of Chemistry, Durham University, South Road, Durham DH1 3LE, UK. Email: [email protected]

species. They have not yet been observed for molecule-molecule collisions, but atommolecule collisions provide a simpler case for study. Son et al. have observed a resonance due to a long-lived state formed in a collision between 23Na atoms and 23Na6Li molecules. They have measured the rate of the reactive atom-molecule collision, NaLi+Na Li+Na2, in the vicinity of this resonance, reporting more than a factor of 100 variation as the magnetic field is tuned across the resonance. To understand the results reported by Son et al., it is useful to consider a simple physical picture of ultracold molecular collisions. As atoms and molecules approach one another from afar, they experience an attractive van der Waals force. At ultracold temperatures, the colliding particles behave as matter waves—a consequence of wave-particle duality in quantum mechanics—and experience partial reflection from the attractive potential. As the colliding particles get closer to each other, the atommolecule pair either remains in the initial state and is reflected off the repulsive core of the potential or is converted to a different state in which chemical reaction can occur (see the figure). If the colliding species are completely lost at short range, the overall loss rate is determined by the quantum reflection of the matter-wave from the attractive potential at long range. However, if the loss at short range is very small, the reflection from the short-range interaction may be followed by a second partial reflection from the attractive potential. This can continue ad infinitum, with multiple reflections back and forth within the molecular potential before the pair separates. Son et al. sought to explain this behavior by adapting the existing model for the reflection of light inside a cavity with partially reflecting mirrors. The light wave escaping from such a cavity interferes with the wave originally reflected from the mirror where the light was first introduced. This interference can be either constructive or destructive depending on the spacing of the mirrors, leading to either enhanced or reduced overall reflection respectively. In the case of colliding particles, 4 MARCH 2022 • VOL 375 ISSUE 6584

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Ultracold chemical reaction using a magnetic field

QUANTUM INFORMATION

Multiple reflections within the intermolecular potential result in quantum interference that strongly affects the chemical reaction. Changing the magnetic field can tune the loss of NaLi molecules during collisions. Weak loss at short range leads to a large range of control in the overall loss rate.

A new Hall for quantum protection

1 The Na atom and NaLi

Magnetic field

to loss through a chemical reaction. Na2

To collision molecule are set to collide.

Li

Na

Reaction

The intermolecular potential is attractive at long range but strongly repulsive at short range.

Collision

2 Interference is controlled

3b Elastic collision occurs

by the magnetic Feshbach resonance.

treated as matter-waves, the presence of a magnetic Feshbach resonance allows the interference to be tuned with the applied magnetic field, akin to changing the spacing between the mirrors. This allows the probability of a reactive collision to be controlled. The long-lived state of the complex formed during the collision that causes the resonance produces very little loss at short range. This is analogous to a highly reflecting inner mirror. The resulting highcontrast interference leads to large changes in the overall loss rate, from far below the universal rate to far above. Using a quantum mechanical scattering model based on an absorbing boundary condition at short range that is analogous to the optical cavity model, Son et al. estimated that only about 4% of the colliding pairs that reach short range are lost, which is far below the 100% loss at short range associated with the universal rate. The much lower loss at short range leads to a large variation in the reaction rate across the resonance. The authors contrast this resonance with a weaker one at a higher magnetic field, where the associated state of the complex is much shorter-lived, which results in a far smaller variation in the reaction rate across the resonance. The authors show that ultracold collisions between Na and NaLi can be understood with relatively simple models. Their experiment provides an excellent testing ground for refining the understanding of ultracold chemistry. Looking to the future, tunable Feshbach resonances may ultimately form the basis of a coherent ultracold chemistry, in which bimolecular reactions are performed coherently across an 976

NaLi

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when the magnetic field is away from Feshbach resonance.

entire sample of molecules. Such a chemistry will allow polyatomic molecules to be built up from their constituent parts while maintaining them all in a single quantum state. This contrasts with the form of coherent control achieved with femtosecond lasers (12), in which coherence exists within a single molecule but not across the entire sample. The ability to control ultracold molecular collisions will benefit many applications. For example, molecules with dipole moments offer fascinating opportunities to study quantum many-body physics in the presence of long-range interactions. This may help understand complex phenomena, such as superconductivity and exotic forms of magnetism. However, achieving this will undoubtedly require the ultracold chemistry to be under control. j REF ERENCES AND NOTES

1. C. Chin, R. Grimm, P. Julienne, E. Tiesinga, Rev. Mod. Phys. 82, 1225 (2010). 2. K.-K. Ni et al., Science 322, 231 (2008). 3. H. Son et al., Science 375, 1006 (2022). 4. P. D. Gregory et al., Nat. Commun. 10, 3104 (2019). 5. R. Bause et al., Phys. Rev. Res. 3, 033013 (2021). 6. P. Gersema et al., Phys. Rev. Lett. 127, 163401 (2021). 7. P. S. Żuchowski, J. M. Hutson, Phys. Rev. A 81, 060703 (2010). 8. M. Mayle, G. Quéméner, B. P. Ruzic, J. L. Bohn, Phys. Rev. A 87, 012709 (2013). 9. A. Christianen, M. W. Zwierlein, G. C. Groenenboom, T. Karman, Phys. Rev. Lett. 123, 123402 (2019). 10. Y. Liu et al., Nat. Phys. 16, 1132 (2020). 11. P. D. Gregory, J. A. Blackmore, S. L. Bromley, S. L. Cornish, Phys. Rev. Lett. 124, 163402 (2020). 12. C. Brif, R. Chakrabarti, H. Rabitz, New J. Phys. 12, 075008 (2010). ACKNOWL EDGMENTS

Long-range vacuum fluctuations break the integer quantum Hall topological protection By Angel Rubio1,2

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avitronics—a portmanteau of cavity and electronics—are devices with certain properties that can be controlled by the light waves bouncing inside the cavity in which the device sits. In quantum mechanical terms, this interaction between light and matter is done by the standing light waves inside the cavity known as vacuum field states. A major advantage of this setup for generating light-matter coupling is the ability to induce certain properties inside a material that otherwise require the use of a strong external electric or magnetic field (see the image). On page 1030 of this issue, Appugliese et al. (1) provide a special case of cavitronics. Their experimental setup modifies one of the most prominent quantum phenomena in materials, known as the quantum Hall effect (QHE). They found a drastic change in its Hall resistance, opening the path to designing materials functionalities by vacuum-field engineering. When an electric current is passed through a conductor under the influence of a magnetic field, an electrical voltage perpendicular to the current is induced by the magnetic field. This is known as the Hall effect. However, this “sideways” voltage sometimes happens in steps rather than linearly as a function of the magnetic field (2, 3). This constitutes the QHE, namely a quantized version of the classical Hall effect (2), in which the Hall conductance exhibits steps, or Hall plateaus, at precisely integer and fractional multiples of the inverse of the quantum of resistance (4). Whereas conventional resistance de1

The authors acknowledge support from the UK Engineering and Physical Sciences Research Council (EPSRC) through grants EP/P01058X/1 and EP/P008275/1. 10.1126/science.abn1053

Max Planck Institute for the Structure and Dynamics of Matter and Center for Free-Electron Laser Science, Luruper Chaussee 149, 22761 Hamburg, Germany. 2Center for Computational Quantum Physics (CCQ), The Flatiron Institute, 162 Fifth Avenue, NY 10010, USA. Email: [email protected] science.org SCIENCE

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To reaction

3a Transmission at short range leads

ILLUSTRATION: JÖRG HARMS AND ANGEL RUBIO/MPSD, HAMBURG

pends on the material’s geometry and exdark cavity, without external illumination, stration that the cavity vacuum field states ternal factors, the quantum Hall resistance breaks the quantization of the Hall conduccircumvent the QHE topological protection depends only on fundamental constants tivity over a wide range of applied magnetic supports recent predictions about the cavand can be reproduced with extraordifields. They measured up to 11 integer Hall ity modification of the quantum Hall resisnary precision. Not surprisingly, the QHE plateaus for the device outside the cavity. tance in defect-free 2D devices (12) when has become the primary standard of reThe shape of those plateaus is modified the cavity field is larger than the external sistance metrology. The robustness of the when immersed in the cavity, with espeapplied magnetic field. QHE to structural defects, disorders, and cially strong changes for odd-numbered Experimental and theoretical research other perturbations is a prominent maniplateaus. The authors also found that the on the control of materials by engineering festation of “topological protection,” a defractional Hall plateaus are much less afcavity vacuum fields is destined to considersirable and advantageous ably accelerate and expand. property for many emergControlling the environing quantum information ment surrounding a mateapplications. Without this rial inside a cavity can alter protection, the quantum its properties, enabling the states of materials are subdesign of quantum matejected to changes caused rials and phenomena (7). by temperature-dependent Recent theoretical predicscattering and dissipative tions for a material’s quanprocesses. To protect certum phenomena mediated tain quantum states from by cavity photons include external disturbances, maphoton-mediated superconterials with specific symductivity, ferroelectricity, metries can decouple some and magnetism, as well as quantum states from interthe control of many-body acting with the rest of the interactions and topological sample. Those are the sophenomena in multilayer called topological protected 2D materials. These are states, which are robust to among the lines of research external perturbations and that may soon come under have long lifetimes. the umbrella of “cavity maHow robust can this topoterials engineering,” which logical quantum protection can be used to induce spebe, with respect to spatially cific functional properties, long-range perturbations Cavitronics are devices whose properties can be controlled with the light waves, or vacuum such as superconductivity, acting on the entire device? field states, of the cavity. Researchers created a cavitronic device embedding a in materials. For example, To answer this question, two-dimensional electron gas in a cavity where the quantized Hall conductivity is modified, it may be possible to conAppugliese et al. created paving the road for future design of materials for quantum technology applications. trol, in twisted bilayer graa two-dimensional (2D) phene and transition metal electron-gas device embedded in a splitfected by the cavity, which seems to be condichalcogenides, the low-energy scales of ring cavity resonator. Vacuum field states, sistent with the observation that fractional their moiré electronic bands and electronwhich span the whole space inside the cavplateaus couple weakly to light (3, 4). Those correlated phases with engineered cavity ity, provide the sought spatially long-range findings offer a new route to control quanvacuum field states to create new exotic interactions inside the device. This interactum materials. states of matter (13, 14), increasing the imtion creates new light-matter hybrid states, Why does the cavity modify the otherwise pressive portfolio of materials phenomena referred to as polaritonic states, with chartopologically protected integer quantum in 2D heterostructures. j acteristics that neither the original mateHall conductivity in quantum Hall devices? REF ERENCES AND NOTES rial nor the cavity field state possesses. This To answer this, it is important to bring up 1. F. Appugliese et al., Science 375, 1030 (2022). hybridization effect is proportional to the the connection between topological pro2. K. Klitzing, G. Dorda, M. Pepper, Phys. Rev. Lett. 45, 494 strength of the light-matter coupling inside tection and the physical path taken by the (1980). 3. I. V. Kukushkint al., Nature 415, 409 (2002). the cavity and can be better detected if the physical Hall current, which is localized at 4. H. L. Stormer, D. C. Tsui, A. C. Gossard, Rev. Mod. Phys. 71, device is brought to a strong coupling rethe edges of the device. The robustness of S298 (1999). gime (5–9), but this is often not the case as the QHE is linked to the physical separation 5. C. Genet, J. Faist, T. W. Ebbesen, Phys. Today 42, (2021). 6. F. J. Garcia-Vidal, C. Ciuti, T. W. Ebbesen, Science 373, the light-matter coupling is generally weak. of this edge current from the bulk of the eabd0336 (2021). By confining the light field in a small resample. However, the vacuum field states 7. H. Hübener et al., Nat. Mater. 20, 438 (2021). gion of space (the cavity), the probability of of the cavity facilitate the hopping of elec8. A. Frisk Kockum et al., Nat. Rev. Phys. 1, 19 (2019). 9. P. Forn-Díazt al., Rev. Mod. Phys. 91, 025005 (2019). photon absorption and therefore the lighttrons between the states carrying the edge 10. G. L. Paravicini-Bagliani et al., Nat. Phys. 15, 186 (2019). matter coupling strength, increases. Recent current and the bulk states of the sample. 11. C. Ciuti, Phys. Rev. B 104, 155307 (2021). advances in cavity design have enabled the This new coupling channel gives rise to the 12. V. Rokaj, M. Penz, M. A. Sentef, M. Ruggenthaler, A. Rubio, arXiv 2109.15075v1 (2021). practical realization of this strong lightbreakdown of the topological protection of 13. L. Balents, C. R. Dean, D. K. Efetov, A. F. Young, Nat. Phys. matter coupling regime (10). the QHE. The microscopic process behind 16, 725 (2020). The device created by Appugliese et al. rethis coupling involves several intermediate 14. D. M. Kennes et al., Nat. Phys. 17, 155 (2021). alizes a strong light-matter coupling regime states in which the defects in the sample Surprisingly, the authors observed that a play an important role (11). The demon10.1126/science.abn5990 SCIENCE science.org

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CATALYSIS

Catalyst coaxed to make branched a-olefins Process conditions allow a titanium catalyst to add just two ethylene molecules to an a-olefin and one a-olefin, and 1-butene. Notably, no polymer by-products were observed inear and branched a-olefins are an that could lead to fouling issues and that important class of chemicals that are plague many ethylene tetramerization catused for making polyolefins, deteralyst systems (9). gents, plasticizers, and lubricants, as This excellent selectivity does not come well as other products, and are made with any sacrifices in catalyst activity, with in quantities that exceed 3.2 million turnover frequencies reported to be as tonnes per year (1). These a-olefins, which high as 2.3 × 106 per hour. Based on the have been produced on an industrial scale catalyst and product amounts in the profor almost a century (2), are typically made vided examples, this rate can be converted in processes that generate a broad distributo a catalyst activity of 2.1 × 106 grams of tion of products. More recently, on-purpose product per gram of titanium per hour, technologies have been developed which is a comparable activity to to synthesize particular linear atop-performing chromium-based olefins. By contrast, branched ethylene trimerization and tetOn-purpose oligomerization a-olefins are produced in nonramerization catalysts (8, 13). The synthesis of a-olefins usually forms a broad product distribution, but selective processes such as fluid Dietel et al. also demonstrate the several selective routes for linear a-olefins have been developed. Dietel et al. catalytic cracking and oligomerversatility of the catalyst system now report that a titanium catalyst run under olefin-rich conditions is highly ization of olefins with acid-based by successfully performing this selective for producing branched a-olefins. or transition metal catalysts (3, trimerization reaction with lin4). On page 1021 of this issue, ear a-olefins from 1-pentene to Linear a-olefins Branched a-olefins Dietel et al. (5) now add to the 1-hexadecene and also with noncollection of on-purpose a-olefin aliphatic and cyclic a-olefins like 1-hexene technologies with their report of 1-butene styrene and norbornene. 1989 1960 a method for selectively syntheThis work enables the selecsizing branched a-olefins. tive synthesis of a broad range of 4-ethyloct-1-ene Dietel et al. 2022 On-purpose technologies for branched a-olefins and undoubt1-octene 2004 linear a-olefin synthesis first apedly will spur additional research peared with the report of ethylin this area. Ultimately, the sysene dimerization to make 1-butene in 1960 a-olefin, the authors produced a branched tem described by Dietel et al. demonstrates (see the figure) (6). Later advances include a-olefin trimer product that incorporates the power of combining both judicious ethylene trimerization to produce 1-hexene two ethylene molecules and one a-olefin catalyst design along with careful choice in 1989 (7) and ethylene tetramerization (see the figure). of process conditions to make an otherwise to produce 1-octene in 2004 (8). The imThe combination of catalyst and process conventional catalyst perform useful reacportance of these processes is reflected in conditions described by Dietel et al. results tions. New products are likely to be develtheir subsequent commercialization, and in up to an impressive 74 mole % selectivoped that take advantage of the availability all three are practiced on an industrial scale ity of the desired branched a-olefin prodof pure branched a-olefins. j today (9, 10). uct. This selectivity is excellent given that, REF ERENCES AND NOTES The impressive selectivities of the titauntil this report, it had been thought that a 1. P.-A. R. Breuil, L. Magna, H. Olivier-Bourbigou, Catal. Lett. nium- and chromium-based catalysts used metallacycle mechanism would be needed 145, 173 (2015). for selective ethylene oligomerization have to explain a selective olefin trimerization 2. C. P. Nicholas, Appl. Catal. A Gen. 543, 82 (2017). 3. Y. V. Kissin, Catal. Rev., Sci. Eng. 43, 85 (2011). been attributed to a mechanism that uses (7, 11). Important to achieving the high 4. A. Forestière, H. Olivier-Bourbigou, L. Saussine, Oil Gas metallacycle intermediates (7, 11). Notably, selectivity in this work are key attributes Sci. Technol. 64, 649 (2009). the catalyst designed by Dietel et al. inof the catalyst: It terminates the growing 5. T. Dietel, F. Lukas, W. P. Kretschmer, R. Kempe, Science 375, 1021 (2022). stead makes use of more traditional transichain readily, and, as a good a-olefin in6. K. Ziegler, H. Martin, US Patent 2,943,125 (1960). tion metal–based ethylene polymerization corporator, it has a high probability for in7. J. R. Briggs, J. Chem. Soc. Chem. Commun. 1989, 674 catalysts, which typically operate through cluding any a-olefins present in the grow(1989). 8. A. Bollmann et al., J. Am. Chem. Soc. 126, 14712 (2004). a Cossee-Arlman mechanism, to produce ing oligomer chain. When such a catalyst 9. O. L. Sydora, Organometallics 38, 997 (2019). a Schulz-Flory distribution of polymers of is combined with a reaction environment 10. D. S. McGuinness, Chem. Rev. 111, 2321 (2011). different lengths (9). Polymer molecular that is rich in a-olefins and lean in ethyl11. T. Agapie, S. J. Schofer, J. A. Labinger, J. E. Bercaw, J. Am. Chem. Soc. 126, 1304 (2004). weight is not only defined by catalyst feaene, the selective synthesis of a branched 12. M. C. Baier, M. A. Zuideveld, S. Mecking, Angew. Chem. a-olefin product results. The side products Int. Ed. 53, 9722 (2014). of this reaction include a branched species 13. S. Kuhlmann et al., J. Catal. 262, 83 (2009). Packaging & Specialty Plastics and Hydrocarbons, The with an internal olefin, an a-olefin tetraDow Chemical Company, 240 Abner Jackson Parkway, Lake Jackson, TX 77566, USA. Email: [email protected] mer product made from three ethylenes 10.1126/science.abo1265

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tures such as transition metal and ligand choice but also by process parameters such as ethylene concentration and reaction temperature (12). The system reported by Dietel et al. uses a titanium-based catalyst with aminopyridinato and imidazolidiniminato ligands that, under typical olefin polymerization conditions, produces a Schulz-Flory distribution of low–molecular weight oligomers. However, by selecting process conditions that feature a very low concentration of ethylene and a high concentration of an

science.org SCIENCE

GRAPHIC: K. FRANKLIN/SCIENCE

By Mari S. Rosen

P OLICY FORUM RESEARCH INTEGRITY

A role for funders in fostering China’s research integrity Despite recent progress, challenges remain By Li Tang

G

iven its sheer scale and growth, China’s scientific research and development (R&D) enterprise can have tremendous effects across the global scientific community in terms of both research quality and research integrity (1, 2). Although issues of research funding and integrity arise in countries around the globe, they are particularly salient in China given the major role of government funding in supporting research and the alarming number of scientific publications by authors affiliated with Chinese institutions that have been retracted. With its pivoting position in knowledge production, the Chinese science grant system is taking a greater role in curbing scientific misconduct and fostering research integrity. Despite this, several barriers remain. The Chinese science grant system plays a major role in supporting basic research, including a huge amount of false science as evidenced by journal retractions. Unlike many counterparts in other countries, many Chinese universities and research institutes are often reluctant to initiate and publicize misconduct in’vestigations. As an emerging science power, Chinese attention toward spotting and stopping misconduct is a rather recent phenomenon. With its voluminous history of print publications, it takes quite a while for the Chinese government and scientific community to process and prioritize certain misconducts to investigate, punish, and deter. PROGRESS Policy formulation The Chinese scientific community has taken proactive stances against academic misconduct and toward fostering responsible research. A series of policy documents, initiatives, and codes on research integrity, which often involve the science grant system, were issued, revised, and adopted across different School of International Relations and Public Affairs, Fudan University, Shanghai, 200433, China. Email: [email protected] SCIENCE science.org

sectors over the past 3 years. For example, in 2019 the Ministry of Science and Technology (MOST), the Central Propaganda Department, the Ministry of Education (MOE), the National Health Commission (NHC), the National Natural Science Foundation of China (NSFC), the Chinese Academy of Social Sciences (CASS), the Ministry of Finance (MOF), and 13 other central agencies jointly issued The Rules for the Investigation and Handling of Research Integrity Cases (Trial). Focusing on research grants management, the revised version of Measures for Investigation and Handling of Scientific Research Misconduct in National Natural Science Foundation of China became effective from 1 January 2021. The most recent and prominent change is demonstrated in the Law on Scientific and Technological Progress of the People’s Republic of China effective on 1 January 2022. This marks the first time that Chinese law (instead of past agency regulation or measures) has prohibited individuals and organizations from buying research papers, grant proposals, and laboratory experiments. In a departure from its 2007 version, research integrity (pinyin, “keyan chengxin”) appeared in six articles of the latest law promoting science, technology, and innovation, laying legal foundation of research integrity cultivation. Implementation In addition to issuing regulations and codes, the enforcement efforts of the Chinese science grant system in curbing misconduct are evident. For example, the NSFC, China’s largest funder of basic research, has adopted an integrated approach to crack down on funding-related misconduct. They limit the number of proposals an individual can submit per, for example, a given time, competition, or agency; restrict the number of supporting representative research publications that can be submitted; use plagiarismdetection software for grant proposals; publicize investigation results on confirmed misconduct of both unfunded grant applications and awarded grants; release names of proposal reviewers; and earmark grants for research integrity studies. The NSFC is not the only agency demon-

strating a strong commitment to research integrity. In June 2021, the NHC launched a special column on its official website, providing education and training on research integrity for medical researchers and practitioners and publicizing misconduct investigation results. Within 7 months (June to December 2021), the NHC released 310 investigation results involving more than 850 researchers with confirmed research misconduct, with punishments ranging from admonishment to disqualification of career promotions and grant applications in given years to suspension and debarment. CASS, the statutory agency overseeing integrity in social and humanities research, launched its official website of research integrity in March 2021. The National Research Integrity Information Management System is in operation and has been compiling information from grantees (affiliations) on proven misconducts, punitive actions, and integrity construction. Evaluation Progress is also underway in how funding proposals are evaluated and performance of awarded grants is assessed, to counteract the detrimental effects of excessive pursuit of publishing in international journals. Research evaluation practices that prioritized tallying articles published in international journals have partially contributed to excessive low-quality articles and false science, which is detrimental to science globally. Encouraging publications in domestic journals, decoupling cash bonuses from publications included in the Science Citation Index (SCI), and shifting emphasis in faculty recruitment selection to teaching and training students are gradually taking place in Chinese elite universities and research institutes. In the merit-review phase of awarded grants, Chinese major administrative apparatus and funding agencies such as MOST, MOE, and NSFC have adopted a one-vote veto of “scientific misconduct” in their funding and talent-rewarding decisions. CHALLENGES The science grant system still faces five barriers that must be dealt with in its ongoing mission of research integrity reform. Dual absence of victim and complainant The damage of research misconduct, although affecting the entire scientific enterprise, is diffused and obscure for specific individuals or groups. Potential direct victims bearing the cost of the violation could be the general taxpayers or grantee’s substitutes (the would-be awardees), who often either do not care or are not aware of this cost. Insiders may remain silent because of con4 MARCH 2022 • VOL 375 ISSUE 6584

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Limited sanction tools of funding agencies Being public organizations, major Chinese science funding agencies such as MOST, NSFC, and MOE have their hands tied with multiple tasks. Curbing misconduct is still in its nascent phase in China. Given the lag of legal authorization and specific measures for punishment, misconduct investigators and funding agencies often use discretionary power to enforce punishments if legal authorization has not been granted. But there is potential risk in doing so. There were some types of fraudulent actions that are not explicitly prohibited on the legal level. Except for explicitly stated limited tools of heightening the scrutiny of grant applications, terminating research funding, retrieving allocated funding, publicizing misconduct investigation, and circulating criticism within their jurisdiction, funding agencies have no direct influence on fraudsters’ salaries, employment, and career development. In the ongoing battle against misconduct, the potential questioning of the legality of available policy tools and punishment actions makes science grant agencies reluctant to exercise penalties. Dilemma of collective punishment Chinese scholars and practitioners remain split on the legitimacy and effectiveness of collective punishment on principal investigators (PIs), affiliated institutions, and team members. Opponents contend that it is unfair for PIs or affiliated organizations to shoulder the responsibility of others’ misbehavior. They believe that an increasing phenomenon of blended or braided funding across different sectors and countries makes collective punishment even more challenging and unrealistic. The supporters, however, argue that credit and responsibility are inseparable from the fault of omission. They hold that responsibility is the price of honor, and liability should not be limited to fraudsters per se. Through collective punishment, the funding agencies could reclaim their monetary losses, remind senior researchers to safeguard the integrity of joint knowledge production, and alert affiliations and research managers of their oversight and education responsibilities. Enforcement difficulties Difficulty of enforcement is a universal challenge in sanctioning infractions, including for misconduct investigations in the Chinese science grant system. Inundated with mount980

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ing grant applications, short-staffed public funding agencies are inclined to support innovative research proposals rather than to oversee, investigate, and sanction fundingrelated misconducts that demand time, financial support, and human resources. Over the period of 2011 to 2020, NSFC funding applications grew at an average annual rate of 9.0%, in sharp contrast to a 3.7% growth rate in the number of in-service personnel at NSFC to handle this increasing workload over the same period. In 2020, the NSFC alone received 281,200 grant applications and funded 45,700 proposals. In the same year, the NSFC initiated 525 misconduct investigations after preliminary review; 165 respondents and eight affiliated organizations were sanctioned to various extents (4).

research paradigms toward team science, interdisciplinary integration, and collaboration involving people of disparate backgrounds.

Classifying gradients of misconducts and degrees of sanctions Chinese funding agencies need to achieve consensus and draft measures to decide

Lack of consensus The progress of research misconduct sanctions and integrity cultivation are imbedded in and thus affected by the national and local cultural, economic, and political systems to which they are oriented. Funding agencies, research managers, and university research ethics committees agree on the direction of the path, but both the pace of development and extent to which institutions can devote resources to the mission remains debatable. Within the Chinese science grant system, different agencies and awardee organizations have not reached an agreement on the demarcation line between research ethics and integrity, questionable research practices and research misconducts, and the punitive scope and severity for principals and accessories involved in proven misconducts. This issue is further compounded by shifting scientific

the gradients of misconducts and degrees of sanctions. Bold policy cannot go far without well-crafted design and solid implementation. Institutionalizing antimisconduct reform, including classifying misbehavior and sanctions for handling alleged misconducts, can help, at least to some extent, to maintain standards even when political focus or media attention wane, to reduce contrived randomness of enforcement when combined with due process and procedural justice for handling alleged misconducts (5), and to make curbing grant-relevant misconducts consistent, manageable, and sustainable across different agencies. Classification needs to take the “first forgive then get tough” principle into consideration (2). To what extent and for how long the limited nonretrospective rule can be applied must be clarified on

ACTIONS These five predicaments are inextricably intertwined and represent great challenges facing the Chinese science grant system to curb misconduct, cultivate integrity, and promote responsible research and innovation. How can funding agencies address or ameliorate these issues? I propose the following suggestions.

science.org SCIENCE

ILLUSTRATION: DAVIDE BONAZZI/SALZMANART

cern about the burden of proof of hard evidence and possible retaliation by fraudsters. This dual absence of victim and complainant signals a low probability of being caught and encourages intentional deviant behavior (3).

the basis of experts’ inputs. This seemingly weak countermeasure may be practical when dealing with the alarming stock of past misconducts and rising retractions of Chinese articles, which could be an artifact of better global oversight or a growing caution toward Chinese contributed knowledge production evidenced by shorter retraction time (6, 7). Strengthening gatekeeping As the engine of basic research and stewardship of public funds, the quality of peer review, anonymous correspondence review, or panel review of grant applications plays a critical role in selection excellence and upholding research integrity. Hometownbased favoritism, rent-seeking, and other nonmerit factors are non-negligible in the Chinese science system (8–10). The RCC (responsibility, credibility, contribution) principle has been recently proposed and trialed by the NSFC (11). However, to be effective in gatekeeping misbehavior, RCC has to be applied in tandem with fair artificial intelligence algorithms to assist grant proposal distribution to a sufficient number of trustworthy and qualified experts. Proper incentivizing Detecting, disclosing, and sanctioning socially deviant behavior can be particularly difficult in Asian cultures that value collectivism and organizational loyalty. To recoup public money misused on fraudulent research, proper incentives need to be adopted for misconduct whistleblowers, expert panels of misconduct investigations, punitive enforcers, and other stakeholders in grant-relevant research activities. Some countries have adopted an appropriate division of recovered grants [for example, in the US case of Thomas vs. Duke University, the court decided the amount of money that Duke is to pay back to the federal government (recovered grant) given its violation of the False Claims Act]. In China, people are rewarded for whistleblowing in some fields such as environmental protection, food and drug safety, and so on. The negative consequences of money for publication may be also applicable to misconduct whistleblowers. Therefore, compensation en masse can be applicable in the science grant system in the future, letting the valid whistleblowers reap the benefits of recovered grants to compensate the risk of retaliation for revealing grant-related frauds. Funding agencies must reward whistleblowers wisely and vigilantly to avoid perverse incentives or bounties that risk glaring instances of alleged misconduct in the pursuit of fortune. Proper incentives should be extended to research managers SCIENCE science.org

and supporting staff for outstanding work in research integrity cultivation. Enhancing transparency Research funded by public money should be available to scientific peers and the public. Chinese funding agencies have made noticeable improvements in transparency aspects, such as NSFC publicizing abstracts of funded proposals and funded research outputs and the National Office for Philosophy and Social Sciences (NOPSS) publishing selected funded research output. NSFC, NOPSS, and more recently NHC regularly disclose grant debarments and investigation results. More can be done. To reduce funding fragmentation and duplicate awards across different agencies (12), the long-called-for integrated funding platform can be extended beyond MOST with awarded projects, research data, and findings of public funded research accessible to the scientific community. The decision by multiple agencies to no longer publicize the names of awardees of prestigious talent programs has raised some concerns within and beyond national borders. More transparency not only reduces reinventing the wheel in public funding and encourages competitive ideas (13) but also alleviates the cost of oversight and deters potential misconduct. Extending integrity education and training It has been increasingly accepted in the Chinese scientific community that integrity education and training outweigh misconduct sanctioning when factoring in effectiveness, efficiency, and equity into consideration. Such education and training need to be more extensive and specialized in the science grant system. The Matthew effect of research resources (“the rich get richer”) combined with the inevitable rise of team science and division of labor calls for team-oriented rather than individual-oriented ethics and integrity training and education (14). Such collaborative education and training should be extended not only to PIs who manage large laboratories and contribute to substantial numbers of publications but also to earlier career researchers, data analysts, and supporting technicians who work in large research teams to be aware of data cross-checking and collaborators’ knowledge validation. In addition, the Chinese science grant system needs to tailor research ethics and academic integrity education considering the differences between natural sciences and social sciences; the latter has been long sidelined in research integrity cultivation. In the evidence-based or evidence-informed

policy-making era, the quality and integrity of social science studies impacts are the same as if not greater than the natural sciences. Extensive and specialized education modules can be set as mandatory before researchers submit grant applications or awardees sign a contract, as well as before midterm assessments for all team members. AGENDA FOR TOMORROW Research integrity is the cornerstone of the science, technology, and innovation system. The Chinese science grant system alone is insufficient to reduce funding-relevant research fraud. In tandem with carefully optimistic and patient objectives, reinforcing research integrity must be advanced in alignment with China’s ongoing reform of science and technology evaluation. It must be systematic and involve orchestrated efforts of different stakeholders across the whole spectrum of scientific exploration, within and beyond national boundaries. For example, Chinese funding agencies can initiate and engage with other stakeholders to participate in and organize global academic conferences and practitioner workshops such as the World Conferences of Research Integrity, Asian and Pacific Rim Research Integrity Network, and so on to share best practices (15). Through people-to-people and agency-to-agency exchanges such as the Integrity Ambassador Program, Chinese funding agencies can strengthen their collaboration with their international counterparts, sending out and inviting in experts to learn from each other to foster research integrity and promote responsible and innovative research. j REF ERENCES AND NOTES

1. National Science Board, National Science Foundation, Science and Engineering Indicators 2020: The State of U.S. Science and Engineering; https://ncses.nsf.gov/ pubs/nsb20201. 2. L. Tang, Nature 575, 589 (2019). 3. E. A. Fong et al., J. Law Med. Ethics 48, 331 (2020). 4. NSFC, NSFC Annual Reports (accessed 2 October 2021); www.nsfc.gov.cn/csc/20345/20290/index. html. 5. C. Hood, O. James, B. G. Peters, C. Scott, Eds., Controlling Modern Government: Variety, Commonality and Change (Edward Elgar Press, 2004). 6. J. Brainard, Science 362, 390 (2018). 7. L. Tang et al., Sci. Eng. Ethics 26, 1681 (2020). 8. Y. Xie, Science 355, 1022 (2017). 9. R. Fisman et al., Polit. Econ. 126, 1134 (2018). 10. Y. Shi, Y. Rao, Science 329, 1128 (2010). 11. G. Chen et al., Bull. Chin. Acad. Sci 36, 1427 (2021). 12. Y. Sun, C. Cao, Science 345, 1006 (2014). 13. S. Feigenbaum, D. M. Levy, Rationality Soc. 8, 261 (1996). 14. J. Walsh et al., Res. Policy 48, 444 (2019). 15. D. Moher et al., PLOS Biol. 18, e3000737 (2020). ACKNOWL EDGMENTS

The research is partially funded by the Ministry of Education (17YJAZH075) and the National Natural Science Foundation of China (L2024009). I thank the four anonymous reviewers for their insightful comments. The views expressed here are those of the author and do not reflect the positions of the funders. 10.1126/science.abm7992 4 MARCH 2022 • VOL 375 ISSUE 6584

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Life-changing biology Genetically engineered genomes will disrupt the bioeconomy and rewrite human nature—are we ready? By Adrian Woolfson

“programmable cells” will, if they are right, eventually upend traditional methods for hen interviewed in 1958 by The building genomes. They assert that we are Paris Review, Ernest Hemingway rapidly approaching a time when it will be confessed to having rewritten the possible to design and artificially syntheending of A Farewell to Arms a size the genomes of living things, including total of 39 times. The writer Raythose of humans, from first principles.  mond Chandler, on the other hand, The release of genomes from the conadopted a different approach. He encouraged straints of evolution and the ability to authors to “throw up” into their typewriters prespecify genetic configurations are, the every morning and “clean up” at noon. authors argue, likely to transform human The strategy adopted by evolution by natunature and that of all living things. At a ral selection in authoring the genomes of livminimum, they may eliminate the “bad ing things has, by necessity, been more aligned genes” underlying straightforward genetic with the latter approach than the diseases and remove some of former. While allowing individual the anguish and uncertainty of letters in the genetic text to be edhuman reproduction. They will ited, and facilitating the deletion, also establish the foundations insertion, recombination, and of a biologically inspired indusduplication of more substantial trial revolution and initiate a genomic regions, evolution predisruptive new bioeconomy. cludes throwing the entire folio DNA sequences might one away and starting again from day replace existing information The Genesis Machine scratch.  Synthetic biology practistorage technologies, the auAmy Webb tioners, however, are not necesthors speculate. Meanwhile, the and Andrew Hessel sarily bound by such constraints. PublicAff synthetic production of protein airs, 2022. 368 pp. In their engaging and endrugs by cells in the body, while ergetic new book,  The Genesis Machine, having a beneficial effect on clinical mediAmy Webb and Andrew Hessel outline an cine, may adversely affect the current biooptimistic manifesto for synthetic biolpharmaceutical industry. And genomically ogy whereby “new biological circuits” and rewritten crops, while helping to achieve global food security, may restructure the agricultural industry. The reviewer is atReplay Holdings LLC, San Diego, CA, USA, Although existing biofoundries and reand author of Life Without Genes (HarperCollins, 2000). Email: [email protected] lated methods of DNA assembly have en-

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abled the construction and recoding of simple genomes such as that of the bacterium Escherichia coli (1), the realization of the type of “genesis machine” envisaged by the authors—an entity capable of synthesizing the genomes of complex organisms from scratch—has remained elusive.  A “DNA typewriter” will be required to achieve this goal. However, given that genome reading technologies have advanced to the point that human genomes can now be sequenced inexpensively within hours, it is not unreasonable to assume that genome writing technologies will follow suit. Even more challenging and timeconsuming than DNA writing is the debugging that synthetic genomes will likely require. Efficient genome writing will need to be tightly coupled to quality control. It is one thing to have the ability to write genomes and another to know what to say. In this regard, databases of DNA sequences derived from natural organisms will help elucidate the principles necessary for effective genome authorship. The success with which machine learning enabled the artificial intelligence program AlphaFold to predict the unknown structures of proteins (2) vindicates this type of approach. Biowarfare and the unpredictability of the ecological impact of bioengineering aside, and regardless of whether we will eventually be forced to choose between extinction or reengineering in order to survive, the issue of who will determine genome authorship, and how it will be deployed and regulated, is nontrivial. The power to control the essential substance of human nature may be a willing handmaiden of totalitarian agendas. Ethical constructs, furthermore, are more fluid than we might like. The authors reference China’s national DNA drive to “collect, sequence, and store its citizens’ genetic data” and its prioritization of synthetic biology in a quest for “global science and tech hegemony.” It appears, however, that governmental agencies have largely ignored the strategic implications of synthetic biology. Without greater attention to this topic, we risk being blindsided by the “great transformation of life” that is already underway. j REF ERENCES AND NOTES

1. J. Fredens et al., Nature 569, 514 (2019). 2. J. Jumper et al., Nature 596, 583 (2021). ACKNOWL EDGMENTS

A.W. is a cofounder of Replay Holdings LLC, which maintains interests in gene therapy, genome engineering, and genome writing. 10.1126/science.abm9852 science.org SCIENCE

PHOTO: JONAS GRATZER/LIGHTROCKET/GETTY IMAGES

A farmer carries freshly harvested genetically modified cauliflower in Panchkhal, Nepal, in 2020.

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SCIENCE LIVES

The lasting legacy of John von Neumann A new biography seeks to reacquaint readers with the once widely celebrated scientist By Dov Greenbaum1,2 and Mark Gerstein2

PHOTO: BETTMANN/CONTRIBUTOR/GETTY IMAGES

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Bhattacharya. “We need you,” the president informed him. The book sketches the natural progression of von Neumann’s academic development, demonstrating how his early work with David Hilbert in Göttingen provided the mathematical foundation for his later work on matrix mechanics and formalizing computers and algorithms and how the degree he earned in chemical engineering from ETH Zurich—a compromise with his banker father, who considered engineering a more financially prudent educational pursuit than mathematics—may have steered him toward more applied pursuits, such as bomb-making

The Man from the Future: The Visionary Life of John von Neumann Ananyo Bhattacharya Norton, 2022. 368 pp.

ohn von Neumann, often referred to by his peers as Johnny, was a Hungarian American polymath and prodigious academic who made vital contributions to numerous fields of study during the first half of the 20th a processing unit, a control unit, random accentury. In 2022, however, von Neumann cess memory, mass storage, and input and could be the smartest person most people output methods. have never heard of. To wit, Google Trends This report was not without controversy. shows that his online popularity last year Von Neumann appears to have scooped the was almost an order of magnitude less efforts of John W. Mauchly and J. Presper than that of Alan Turing, a contemporary Eckert, other early pioneers in the field. More in computing; Erwin Schrödinger, a predethan a mere academic slight, the battle over cessor in quantum mechanics; the invention of the modern comand Stephen Wolfram, a succesputer became “the longest trial sor in the world of automata. in the history of the federal court This remarkable scholar system.” Ultimately, however, the was not always uncelebrated. courts ruled, as von Neumann “When he died, aged just fiftyhad intended, that the basic ideas three, von Neumann was as of the digital computer were left famous as it is possible for a to the public domain. mathematician to be,” writes The reader learns much about Ananyo Bhattacharya in The math and physics in this biogMan from the Future, which exraphy, including the differences amines the tremendous impact between the matrix and wave von Neumann had on various interpretations of quantum mescientific disciplines in eight chanics, Gödel’s incompleteness exceptional chapters. theorems, and the mathematics Bhattacharya observes that that underlies the Turing mavon Newman “constantly sought chine. In contrast, we learn sparnew practical fields to which ingly little about von Neumann’s he could apply his mathematipersonal life: for example, that cal genius, and he seemed to he had an interesting custody choose each one with an unerrVon Neumann receives the Medal of Freedom from President Eisenhower in 1956. agreement with his first wife (his ing sense of its potential to revdaughter, Marina, was to live olutionize human affairs.” His research and and computer engineering. Meanwhile, his with her mother until she reached “the age insights were as invaluable to the emerging work in digital computing led von Neumann of reason,” at which point she would live with military-industrial complex as they were to a late-in-life interest in artificial life, auher father in order to “receive the benefit of to IBM, and his adherents ranged from tomata, and the brain. his genius”), that he enjoyed partying, and hardcore “cold warriors” to groundbreakCompared with his other pursuits, von that he “showed signs of obsessive-compuling computer engineers to radical innovaNeumann’s path to game theory was not sive disorder.” “A drawer could not be opened tors working on artificial life. At the end of as obvious. It is, nonetheless, where he unless it was pushed in and out seven times,” his life, as he lay dying in a hospital, he was made some of his greatest contributions. according to his second wife. “The same with surrounded by the secretary of defense; the “A half-dozen Nobel laureates are recka light switch, which also had to be flipped deputy secretary of defense; the secretaroned to have been influenced by the work,” seven times before you could let it stay.” ies of the air force, army, and navy; and the notes Bhattacharya. Many of von Neumann’s prescient ideas chiefs of staff. He left the hospital briefly in Apart from game theory, von Neumann still play an immeasurable role in modern life a wheelchair to accept the Medal of Freeis perhaps best known today for his work in more than half a century after his death. Redom from President Eisenhower, notes computing. His incomplete First Draft of a acquainting modern readers with “the most Report on the EDVAC (the electronic discrete famous scientist in America after Einstein,” The reviewers are at 1Zvi Meitar Institute for Legal variable automatic computer) has become The Man from the Future calls attention to Implications of Emerging Technologies, Radzyner the “most influential document in the history a conundrum: Why has von Neumann so Law School, Reichman University, Herzliya, Israel; and of computing,” describing what we now intuit largely faded from contemporary view? j 2 Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA. Email: [email protected] as the necessary components of a computer: 10.1126/science.abn7018 SCIENCE science.org

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LET TERS Greece has banned the construction of new roads in some of its mountainous areas, a policy that can proactively protect vulnerable habitats and species.

Greek roadless policy: A model for Europe On 18 January 2022, the Greek government banned road construction in six Natura 2000 roadless mountainous areas, triggering a broader national roadless policy (1). Road sprawl is a key catalyst of land use change, which is the greatest threat to biodiversity worldwide (2). For years, scientists have called for halting infrastructure expansion, including roads, to protect biodiversity (3–6), with little success. Greece’s policy should serve as a model for the EU. Europe is the most road-fragmented continent (4). However, the EU has not yet passed legislation to protect its natural ecosystems from new road construction, as the United States did for its forests in 2001 (5, 6). In 2011, the EU set a goal to eliminate new land conversion to artificial land by 2050 (known as the “no net land take” milestone) (7), but the target was not legally binding. In the years since, land conversion to artificial land, soil imperviousness, landscape fragmentation, and land use change have increased, and terrestrial biodiversity has continued to decline (8). The EU’s 2030 Biodiversity Strategy commits to legally protect a minimum 984

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of 30% of the EU’s land area but does not address road fragmentation (9). The plan will allocate billions of euros for planting trees and restoring degraded environments but leave natural ecosystems vulnerable to new roads and landconsuming projects. This conflict is particularly pronounced in biodiversity-rich European countries such as Spain and Greece, which are facing a rapid expansion of renewable energy infrastructures in ecologically sensitive zones (10, 11). Europe is a densely populated continent that still holds large tracts of natural unfragmented lands (12). The EU should adopt a roadless policy (5) similar to the Greek legislation. Roadless areas should be integrated into the criteria for expansion, interconnection, and strict protection of parts of the Natura 2000 network, and roadless areas should be considered proactive restoration targets (9). The Greek model could provide an efficient, straightforward, and costeffective approach to tackle both climate change and the biodiversity crisis in the EU. Vassiliki Kati1,2*, Nuria Selva2,3 Per Sjögren-Gulve2 1

University of Ioannina, Department of Biological Applications and Technology, GR-45110 Ioannina, Greece. 2Society for Conservation Biology Europe Section, FR-75013 Paris, France. 3Institute of Nature Conservation, Polish Academy of Sciences, 31-120 Kraków, Poland. *Corresponding author. E-mail: [email protected]

REF ERENCES AND NOTES

1. Biodiversity Conservation Lab, University of Ioannina, “Roadless” (2022); https://bc.lab.uoi.gr/en/ research/projects/roadless/. 2. S. Díaz et al., Science 366, eaax3100 (2019). 3. W. F. Laurance et al., Nature 513, 229 (2014). 4. P. L. Ibisch et al., Science 354, 1423 (2016). 5. V. Kati et al., Biol. Conserv. 252, 108828 (2020). 6. N. Selva et al., Environ. Manage. 48, 865 (2011). 7. European Union, “Science for environmental policy. Future brief: No net land take by 2050?” (2016); https://ec.europa.eu/environment/ integration/research/newsalert/pdf/ no_net_land_take_by_2050_FB14_en.pdf. 8. European Environmental Agency, “The European environment-state and outlook 2020: Knowledge for transition to a sustainable Europe” (2019); www.eea. europa.eu/soer/2020. 9. European Commission, “EU Biodiversity Strategy for 2030” (2020); https://ec.europa.eu/environment/ nature/biodiversity/strategy/index_en.htm. 10. D. Serrano et al., Science 370, 1282 (2020). 11. V. Kati et al., Sci. Tot. Environ. 768, 144471 (2021). 12. European Environmental Agency, “Landscape fragmentation pressure and trends in Europe” (2021); www.eea.europa.eu/ data-and-maps/indicators/mobility-andurbanisation-pressure-on-ecosystems-2/ assessment. 10.1126/science.abo2014

Iran’s agricultural waste Global agricultural waste poses a serious threat to food security (1). Each year, Iran wastes 35 million tons of food in different harvesting stages (2), 25 times more than the annual agricultural waste produced by developed countries (3). The wasted products in Iran could feed 15 science.org SCIENCE

PHOTO: HARITAKIS PAPAIOANNOU

Edited by Jennifer Sills

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million people annually (3). The value of the wasted food is equal to the value of half of the country’s oil revenue (4). By wasting food, Iran is also wasting the 300 million liters of fuel and the 13.7 billion cubic meters of water that were required to produce it (1). Because of drought conditions and a climate with three times the global average evaporation rate (1), Iran devotes 90% of its water consumption to agriculture. Iran must mitigate food waste for the sake of food security, water sustainability, and human safety (1). Iran’s inadequate farming practices contribute to food waste. Farmers lack necessary knowledge about the benefits of modern agricultural techniques (2) and productive cultivation patterns (5). Because of rising and unregulated prices (6), many farmers cannot afford modern agricultural equipment (3), packing techniques, warehousing systems, or refrigeration rooms (1). Some waste occurs because crops are not purchased, transported (1, 2, 4, 5), or distributed efficiently (5, 7). Products such as watermelons, tomatoes, and apples are particularly vulnerable to weaknesses in the supply chain (7). Insufficient government oversight allows interference by middlemen (5, 7), which can lead to the production of more supply than demand and the excess produce, such as oranges (7), going to waste. Consumers also contribute to waste. Government subsidies on some products incentivize buying more food than one can eat (1). A range of factors influence how likely consumers are to let food go to waste (8), and current government policies do not take this information into account. Iran’s government could reduce agricultural waste by improving farming standards and providing better incentives. Legislation should be passed to manage import and export of crops (4, 5), guarantee purchase of products from farmers (3), reduce transaction costs (1), and provide agricultural insurance (3). Farmers should receive more support to fight pests and plant diseases, modernize farming equipment (6), provide efficient cold storage of raw products (7), and develop new methods of warehousing such as dynamic and smart atmosphere control systems, advanced ventilation, and online control (1). Small farmers should receive financial support for setting aside cultivation to prevent production that is in excess of consumption (9). The government should invest in the development of new technologies for protecting and harvesting agricultural SCIENCE science.org

products more safely. Some agricultural waste should be used as organic fertilizer, clean fuels, and bio(nano)materials (10). Beyond government, broad national mass media coverage should emphasize the proper consumption of agricultural products. Finally, nongovernmental organizations should spearhead campaigns to encourage food-waste reduction. Reza Akbari1 and Mahmoud Nasrollahzadeh2* 1

Department of Chemistry, Faculty of Science, Gonbad Kavous University, Gonbad, Iran. 2 Department of Chemistry, Faculty of Science, University of Qom, Qom, Iran. *Corresponding author. Email: [email protected] REF ERENCES AND NOTES

1. A. Mirmajidi-Hashtjin et al., “Post-harvest loss reduction: Most important strategic approach in enhancement of food security,” Ministry of Agriculture, Jahad, Agricultural Research, Education, and Extension Organization (2016); http://fipak.areeo.ac.ir/site/ catalogue/18823032 [in Farsi]. 2. “Management and processing of agricultural waste in Iran is necessary,” Science News Agency (2021); https://isfahan.sinapress.ir/news/115076 [in Farsi]. 3. “35% of agricultural products are turned into waste,” Donya-e-eqtesad (2020); www.donya-e-eqtesad. com/fa/tiny/news-3707031 [in Farsi]. 4. M. R. Jamshidi, “Why hasn’t an agriculture reduction research institute been established?,” Food Security Scout News Station (2020); http://iwo.ir/j09rn [in Farsi]. 5. “When the wages of their labors are destroyed by this waste,” Iranian Students News Agency (2020); www. isna.ir/news/99091510891/ [in Farsi]. 6. “Farmers cannot afford to use up-to-date equipment,” Donya-e-eqtesad (2020); www.donya-e-eqtesad. com/fa/tiny/news-3682985 [in Farsi]. 7. M. Naji, “Thirty percent of waste in the cycle of production and distribution of agricultural products,” Broadcasting News Agency of the Islamic Republic of Iran (2021); www.iribnews.ir/00CzMf [in Farsi]. 8. J. Aschemann-Witzel, Sci. Transl. Med. 352, 408 (2016). 9. “The need to use the correct consumption pattern to reduce agricultural waste,” Iranian Students News Agency (2013); www.isna.ir/news/92070603338/ [in Farsi]. 10. C. Xu, M. Nasrollahzadeh, M. Selva, Z. Issaabadi, R. Luque, Chem. Soc. Rev. 48, 4791 (2019). 10.1126/science.abn9765

China’s dams threaten the Sichuan taimen The Sichuan taimen (Hucho bleekeri), a glacial relict and freshwater salmonid fish endemic to the Yangtze River (1), has been categorized as Critically Endangered on the Red List of the International Union for Conservation of Nature (2) and is listed as an endangered species on China’s Species Red List (3). There are currently between 2000 and 2500 Sichuan taimen individuals, and the population is likely to decrease by more than 20% over the next two generations (2, 4). The species’ decline is the result of

habitat loss, overfishing, pollution, and climate change (2, 5–7). The planned construction of the Barra dam now threatens to destroy the Sichuan taimen’s last refuge (8, 9). China has decided to invest a sum equivalent to about US$63.4 billion in hydropower along the Yangtze River (10), where the Sichuan taimen lives. Four massive dams are planned to help China decrease its reliance on fossil fuel energy (10). Hydropower projects have already reduced the habitats of these fish from 5000 km2 in the 1960s to less than 100 km2 (11). The Barra hydropower station will destroy the four remaining Sichuan taimen spawning sites (8). Without these important habitats, the species may die out. A survivor of the quaternary glacial period, the Sichuan taimen is a valuable genetic resource with scientific value for studying animal geography, paleoecology, and climate change (11, 12). To protect the Sichuan taimen, the Chinese government should understand the risks of its extinction and should designate the spawning sites as natural reserves. The construction of the Barra hydropower station should stop until a sustainable path forward for the Sichuan taimen is found. Meanwhile, China should implement scientific management and protection policies to minimize habitat fragmentation and illegal fishing in the Yangtze River. Di Tong College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China, and Zhejiang Provincial Key Laboratory of Agricultural Resources and Environment, Zhejiang University, Hangzhou 310058, China. E-mail: [email protected] REF ERENCES AND NOTES

1. K. Wang et al., J. Appl. Ichthyol. 32, 343 (2016). 2. Z. Song, “Hucho bleekeri (errata version published in 2020).” The IUCN Red List of Threatened Species (2012); www.iucnredlist.org/ species/13151680/174797529. 3. Y. Wang et al., Mar. Genom. 4, 221 (2011). 4. P. S. Rand, Arch. Polish Fish. 21, 119 (2013). 5. G. Tong et al., Aquaculture 529, 735680 (2020). 6. Y. Chen et al., Dev. Compar. Immunol. 116, 103934 (2021). 7. M. Hu, Y. Wang, L. Cao, B. Xiong, Environ. Biol. Fish. 82, 385 (2008). 8. L. Hu,“The 8.15-billion-yuan project threatens Sichuan taimen, a giant panda in the water,” Thepapernet (2021); https://m.thepaper.cn/baijiahao_16083356 [in Chinese]. 9. “Critically endangered Sichuan taimen crisis: The last homes may be destroyed by a hydropower station,” Baidu (2018); https://baijiahao.baidu.com/s?id=1619 645349684424461&wfr=spider&for=pc [in Chinese]. 10. J. Qiu, Science 336, 288 (2012). 11. Y. Zhang, P. Luan, G. Ren, G. Hu, J. Yin, Ecol. Evol. 10, 1390 (2020). 12. Y. Chen et al., Conserv. Genet. Resourc. 12, 157 (2020). 10.1126/science.abo0354 4 MARCH 2022 • VOL 375 ISSUE 6584

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rules affecting transcription, resulting in predictable changes within the genome. —LMZ

RESEARCH

Science, abg0162, this issue p. 1000

TOPOLOGICAL PHYSICS

A monopole set in diamond

IN S CIENCE JOURNAL S

Magnetic monopoles, hypothetical sources of magnetic field in three-dimensional space, have not been observed as elementary particles. However, synthetic monopoles can be engineered in ultracold systems. Going a step further, Chen et al. used the quantum levels of a nitrogen vacancy center in diamond to observe the effects of a synthetic tensor monopole: the generalization of the magnetic monopole to four dimensions. The researchers modulated an applied microwave pulse to measure the “magnetic” field emanating from the tensor monopole and its topological charge. —JS

Edited by Michael Funk

Science, abe6437, this issue p. 1017

MOLECULAR KNOTS

Opportune mismatches The typical approach to making molecular knots is to use metal ions to orient ligand strands in mutually overlapping arrangements. Ashbridge et al. added a Vernier templating technique to expand the size and complexity of accessible knots. Specifically, they introduced a mismatch between the numbers of coordination sites on the ligands and on the metal ions, resulting in an assembly comprising the lowest common multiple of sites. A 3:4 stoichiometry produced a 378-atom trefoil-of-trefoils after ring closure by olefin metathesis. —JSY

KIDNEY DISEASE

Fingering FANCD2 in tubular repair

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he transition from acute kidney injury, characterized by intrinsic repair, to incomplete repair and chronic damage has been difficult to study. Gupta et al. modeled the transition from intrinsic to incomplete repair using human kidney organoids. A single exposure to cisplatin resulted in intrinsic repair, with preserved tubular architecture and up-regulation of genes associated with homology-directed repair, including Fanconi anemia complementation group D2 (FANCD2). However, with repeated cisplatin exposure, FANCD2 and RAD51 recombinase (RAD51) were down-regulated, leading to incomplete repair. The DNA ligase IV inhibitor SCR7 increased FANCD2-mediated repair and ameliorated progression to chronic injury in the organoids, suggesting that targeting the FANCD2/RAD51 pathway may have the potential to treat kidney disease. —MN Sci. Transl. Med. 14, eabj4772 (2022). Fluorescence microscopy image of proximal tubules in a kidney organoid that have been immunostained to detect DNA damage markers (green)

SYNTHETIC GENOMICS

IMAGE: GUPTA ET AL.

Predicting effects from genomic changes Genetic modifications of organisms can create artificial, synthetic genomes. Such changes are forecast to be useful for understanding the principles SCIENCE science.org

of how genomic context affects cellular function. However, the effects of most drastic genomic movements are unknown, never having been observed in nature. Brooks et al. examined a line of yeast that are easily induced to move genes within the genome to determine how the context of

genes affects their expression. Examining 612 genetic changes, the authors found differences in the length and content of transcripts, which indicates that the local genetic environment is important for transcription. From these experiments, the authors were able to determine specific

Science, abm9247, this issue p. 1035

EPIGENETICS

Safeguarding stem cells Bivalent genes co-occupied by the epigenetic markers histone 3 trimethyllysine 4 (H3K4me3) and 27 (H3K27me3) were first

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Science, abm0730, this issue p. 1053

ASTEROIDS

Obtaining and returning samples of Ryugu The Hayabusa2 mission investigated the nearby carbonaceous asteroid Ryugu and collected samples for return to Earth. Tachibana et al. describe Hayabusa2’s second sample collection, which picked up material that was excavated from Ryugu’s subsurface by an earlier impact experiment. By analyzing footage from both touchdown events, the authors determined the morphological properties of small pebbles on the asteroid. After the sample return capsule was opened on Earth, they found that

A small pebble returned from the asteroid Ryugu to Earth by the Hayabusa2 mission

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the chambers contained pebbles with properties consistent with the touchdown locations, indicating that they are representative samples of Ryugu’s surface and subsurface. —KTS

IN OTHER JOURNALS

Edited by Caroline Ash and Jesse Smith

Science, abj8624, this issue p. 1011

CORONAVIRUS

Omicron’s defenses and vulnerabilities The Omicron variant of severe acute respiratory syndrome coronavirus 2 quickly became dominant across the world partly because of its ability to evade existing immune defenses. Yin et al. provide structural data showing how the Omicron spike protein maintains and even strengthens binding to the human ACE2 receptor, whereas binding to most existing therapeutic antibodies is inhibited. JMB2002, an antibody that has completed phase 1 clinical trials, effectively neutralizes Omicron as well as variants Alpha, Beta, and Gamma, but not Delta. A structure shows the basis for this breadth. —VV Science, abn8863, this issue p. 1048

PLANT ECOLOGY QUANTUM SIMULATION

Time crystal in quantum computer One of the most promising applications of quantum computers is to simulate quantum phenomena that are difficult to efficiently simulate using classical computers. A time crystal is a unique quantum phase of matter that repeats in time and never reaches thermal equilibrium. Frey and Rachel used a state-of-the-art quantum computer involving 57 qubits to realize this state, and their experiments show that the true dynamics of a discrete time crystal appear in the quantum computer. This study highlights the power of a quantum computer to explore quantum phenomena for sizable quantum systems. —RO

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Sci. Adv. 10.1126/ sciadv.abm7652 (2022).

Island seed dispersal

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adagascar’s large fruit eaters, including elephant birds and giant lemurs, went extinct about 1000 years ago. As a result, seed dispersal for some large-fruited plant species is inefficient. The modern distributions of some plant species may therefore reflect past distributions of extinct animals. Méndez et al. examined the roles of environmental conditions and extinct and extant frugivores on the occurrences of 190 palm species in Madagascar. Living frugivores, including birds and lemurs, had the strongest influence on palm composition across the island. However, palm distributions still showed a small but distinct signature of extinct megafauna dispersal. —BEL Ecography 10.1111/ecog.05885 (2022). Extinct giant lemurs helped distribute the fruit of extant palm trees in Madagascar.

SOCIOLOGY

Developing infant brains by reducing poverty The Baby’s First Years study is a randomized controlled trial that investigates whether

poverty reduction can cause noticeable changes in children’s early brain development. After giving either $333 per month or $20 per month to 1000 low-income mothers from four different metro areas science.org SCIENCE

PHOTOS: (LEFT TO RIGHT) TACHIBANA ET AL.; WORLD HISTORY ARCHIVE/ALAMY STOCK PHOTO

observed in embryonic stem cells (ESCs). Gene bivalency has been widely thought to be a priming mechanism to facilitate gene induction kinetics during differentiation. However, loss of H3K4me3 at bivalent promoters by deleting the methyltransferase Mll2 has little effect on gene induction kinetics. Zhang et al. report that the protein BEND3 is required for optimal H3K27me3 occupancy at hundreds of bivalent promoters, and deletion of the Bend3 gene in ESC causes premature activation of bivalent genes and the failure of differentiation. The authors propose BEND3-mediated Polycomb stabilization as a mechanism to prevent premature activation and to safeguard the differentiation process. —DJ

SYMBIOSIS

Personalized fungal detox

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acteria in the wild use a slew of small molecules to help them earn a living. Phenazines are redoxactive bacterial products that are widespread in the environment and are harmful to fungi but not to all bacteria. Dahlstrom and Newman found in cropland soils that the bacterium Paraburkholderia edwinii protected cohabiting Aspergillus fungi from the toxic effects of phenazine-1-carboxylic acid (PCA). Aggregates of bacteria within a colony of Aspergillus soaked up the PCA, whereas the fungus promoted acidic reducing conditions that limited the toxicity of the sequestered PCA. Aggregation in P. edwinii is regulated by the transcriptional repressor HrcA, which is found in many bacteria with fungal partners. HrcA could be as important in establishing interkingdom microbial communities on coral reefs or in the human gut as it is in the rhizosphere. —CA Curr. Biol. 32, 275 (2021). The fungus Aspergillus finds refuge from toxins within colonies of the bacterium Paraburkholderia edwinii.

in the United States, the study reports statistically significant differences in the electroencephalography brain scans of 1-year-old infants from the two groups. The study is ongoing and will follow these families to determine whether the observed differences between the two groups will persist and lead to cognitive and behavioral differences later on. —YY

complexities of the unfolded protein response signaling in mouse models, then attempted to deflect the response using a small-molecule compound. The treatment improved glucose metabolism and reduced the accumulation of fat in the animals’ livers, indicating its potential for therapeutic applications. —YN Nat. Commun. 13, 608 (2022).

However, immunogenic tumor neoantigens from human melanoma and lung cancer mostly have intermediate TCR affinity. Subsequent fine-tuning of the TCR signal to the intermediate “sweet spot” improved the ability of T cells to elicit tumor cell clearance and may provide avenues for improved cancer immunotherapies. —PNK

of homogeneous catalysts and could be adapted to other classes of ligands in the future. —YS

J. Exp. Med. 219, e20201966 (2022).

Gender wage gaps may have roots in men being more selfpromoting than women. Exley and Kessler had more than 4000 adults perform tasks and then rate their subjective performance on a scale. Men judged their math and science performance more favorably than did equally performing women regardless of whether participants were first told how they actually scored and if they were first told that the selfevaluations were to be shared with potential employers or kept private. Men and women rated performance similarly for other participants and for themselves on a test of verbal skills. Similar gaps were seen in more than 10,000 middle- and high-school-aged participants. —BW

Proc. Natl. Acad. Sci. U.S.A. 119, e2115649119 (2022).

OBESITY

IMAGE: KURT DAHLSTROM

Unfolding the sequelae of obesity Obesity and type 2 diabetes are increasingly common worldwide and are associated with numerous health complications. Given the complexity of these metabolic illnesses, it is not surprising that multiple signaling pathways are involved and the connections are not always straightforward. For example, activation of the unfolded protein response can be protective or detrimental depending on the degree and duration of activation. Madhavan et al. have untangled some of the SCIENCE science.org

CANCER IMMUNOLOGY

MACHINE LEARNING

Sweet spot for TCR

Exploring organophosphorus ligands

The T cell receptor (TCR) is a complex on the surface of T lymphocytes that participates in the activation of antitumor T cell immune responses. During acute infections, high-affinity interactions with pathogen antigens trigger T cell expansion, but in cancer, TCR affinity to tumor self-antigens may be low. Shakiba et al. examined how different TCR signal strengths affected the ability of a subset of T cells called CD8+ T cells to control tumors in mice. Weak TCR–tumor antigen interactions led to ineffective cancer cell killing, whereas strong TCR–tumor antigen engagement drove T cells into a nonfunctional state.

Machine learning (ML) has become a mainstream tool in the development of numerous algorithms for virtual chemical space exploration and is increasingly being applied to more and more complex chemical systems. Gensch et al. present a comprehensive ML-based discovery platform to study the property space of organophosphorous (III) ligands (actively used in homogeneous catalysis), which was able to predict 190 physicochemical descriptors for more than 300,000 new ligands. Their platform could be used for the inverse design

J. Am. Chem. Soc. 144, 1205 (2022).

GENDER GAP

The manly art of self-promotion

Q. J. Econ. https://doi.org/ 10.1093/qje/qjac003 (2022). 4 MARCH 2022 • VOL 375 ISSUE 6584

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ALSO IN SCIENCE JOURNALS GENETICS

Cell type diversity in a whole fly The fruit fly Drosophila melanogaster has served as a premier model organism for discovering fundamental and evolutionarily conserved biological mechanisms. Combining recent advances in single-cell sequencing with powerful fly genetic tools holds great promise for making further discoveries. Li et al. present a single-cell atlas of the entire adult fruit fly that includes 580,000 cells and more than 250 annotated cell types. Cells from the head and body recapitulated cell types from 15 dissected tissues. In-depth analyses revealed rare cell types, cell-type-specific gene signatures, and sexual dimorphism. This atlas provides a resource for the Drosophila community to study genetic perturbations and diseases at single-cell resolution. —BAP Science, abk2432, this issue p. 991

NEUROGENOMICS

Mapping the brain’s blood vessels Cerebrovascular diseases are a leading cause of death and disability, but our understanding of the cellular and molecular constituents of normal and diseased human cerebrovasculature is incomplete. Winkler et al. generated a cellular-resolution atlas of the adult human cerebrovasculature. The authors used spatial transcriptomics to reveal the geographical organization of an unexpectedly diverse array of molecularly defined cell types within the human brain. They then explored the cellular and molecular alterations that occur in arteriovenous malformations, a leading cause of stroke in young people. A specialized subtype of peripheral monocyte plays a role in destabilizing the cerebrovasculature, and the 989-B

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authors identified candidate targets for therapeutic intervention. —STS Science, abi7377, this issue p. 992

PLANT SCIENCE

Getting to the root of root development Plants have the useful ability to grow new pieces on an as-needed basis. Omary et al. analyzed the developmental programs that control root development in the tomato plant (see the Perspective by Shaar-Moshe and Brady). Underground, lateral roots are initiated from pericycle tissue of the primary root and branch off from the main root column. Lateral roots can also develop above ground, initiated off of the aboveground shoot. These shoot-borne roots grow from cells associated with the phloem that take on a stem-cell-like state regulated by the transcription factor SHOOTBORNE ROOTLESS (SBRL). Although belowground lateral roots and aboveground shoot-borne roots differ in details, their development shares a suite of transcription factors that regulate a transitional cellular state. —PJH Science, abf4368, this issue p. 993; see also abo2170, p. 974

NEUROSCIENCE

Dopamine and the gating of REM sleep Sleep is composed of rapid eye movement (REM) and non-REM sleep, and REM sleep usually appears after periods of non-REM sleep. However, we do not understand the mechanisms by which the brain cycles between these states. Using fiber photometry, Hasegawa et al. found increases in dopamine activation before non-REM to REM transitions but not before non-REM to wake transitions in the basolateral amygdala (see

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the Perspective by Arrigoni and Fuller). This effect was mediated by dopamine receptor D2-expressing neurons in the amygdala. Artificially activating these neurons induced a transition from non-REM to REM sleep and cataplectic states in awake mice. —PRS Science, abl6618, this issue p. 994; see also abo1987, p. 972

ULTRACOLD CHEMISTRY

Magnetic control of chemistry There is a considerable interest in realizing control of molecular reactions at ultracold temperatures because of unprecedented opportunities in the ultimate quantum regime. Son et al. discovered an ideal system, a sodium atom and a sodium–lithium molecule, in which the combination of Feshbach resonances and low short-range loss probability allows for the significant suppression or enhancement of reactive collisions controlled by external magnetic fields (see the Perspective by Cornish and Hutson). This work presents a method for manipulating the reactivity of ultracold molecules that is qualitatively different from previous experiments. At the same time, this method is highly dependent on the distinctive short-range properties offered by the sodium/sodium– lithium complex and is subject to further testing in other systems. —YS Science, abl7257, this issue p. 1006; see also abn1053, p. 975

CATALYSIS

Ethylene extensions Ethylene is the most common building block of plastics, but it is often mixed with longer carbon chains during polymerization to tune material properties. Selective preparation of particular longer, terminal olefins is therefore an area of active

research. Dietel et al. report that a titanium catalyst with optimized nitrogen-coordinating ligands can add two equivalents of ethylene to a terminal olefin, extending the main chain by two carbons while also appending an ethyl branch (see the Perspective by Rosen). The authors attribute the selectivity to unusually rapid b-hydride elimination. —JSY Science, abm5281, this issue p. 1021; see also abo1265, p. 978

MAGNETISM

Magnons in a skyrmion lattice Electrons in two-dimensional solids placed in an external magnetic field fill the so-called Landau energy levels. In materials with a nontrivial spin texture, spin excitations (magnons) may have an analogous energy-level structure. However, showing this effect in an experiment is tricky. Weber et al. used inelastic neutron scattering and numerical simulations to demonstrate this effect in the skyrmion lattice phase of the material manganese monosilicide. —JS Science, abe4441, this issue p. 1025

QUANTUM OPTICS

Harnessing vacuum fluctuations Vacuums are not simply empty voids but rather volumes teeming with electromagnetic vacuum fluctuations. Although energy conservation forbids any process that could extract energy from such states, Appugliese et al. show that the enhancement of vacuum field fluctuations in subwavelength split-ring resonators affects the quantum Hall electron transport in high-mobility twodimensional electron gases (see the Perspective by Rubio). Both the longitudinal and transverse values of the resistance depart greatly from their quantized science.org SCIENCE

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values despite the topological protection offered by the quantum Hall effect. These results suggest that vacuum fluctuations could be engineered to optoelectronically control electronic systems without the need for illumination. —ISO Science, abl5818, this issue p. 1030; see also abn5990, p. 976

Kutzner et al. identified an inhibitory phosphorylation site that restrained antigen receptor–induced formation of the CBM complex and lymphocyte activation. This phosphorylation event prevented T cell activation without co-receptor stimulation and sensitized B cell lymphomas to inhibitors used to treat this cancer type. —WW Sci. Signal. 15, eabk308 (2022).

CORONAVIRUS

Enhancing the immune response Many lives have been saved by the vaccines that were rapidly developed against severe acute respiratory syndrome coronavirus 2. As the pandemic continues, we are faced with waning vaccine-induced immunity and variants of the virus that partly evade this immunity, so there are questions regarding boosting strategies. Kaku et al. examined the immunity induced by either prime and boost with the adenoviral vectored vaccine ChAdOx1 or prime with ChAdOx1 and boost with a messenger RNA (mRNA) vaccine. Whereas ChAdOx1 expresses wild-type viral spike, the mRNA vaccines express spike stabilized in the prefusion conformation. Boosting with the mRNA vaccine focuses the response on epitopes in the prefusion conformation and results in overall higher neutralizing activity and increased breadth against variants of concern. —VV Science, abn2688, this issue p. 1041

INNATE LYMPHOID CELLS

Friendly competition in the liver Hepatotropic viruses such as hepatitis B virus (HBV) can cause chronic liver disease in part by generating antiviral effector T cells that subsequently contribute to collateral tissue damage. Using intravital microscopy in mouse models of HBV infection, Fumagalli et al. demonstrate that group 1 innate lymphoid cells (ILCs), comprising natural killer cells and ILC1s, interact closely with HBVspecific CD8+ T cells in the liver and protect against liver damage caused by effector T cells. Rather than directly killing nearby CD8+ T cells, group 1 ILCs limited effector T cell responses by competing for local interleukin-2 required for their proliferation. These results provide insight into cross-talk between liver lymphocyte populations and identify a regulatory function for group 1 ILCs in constraining antiviral T cell responses. —CO

their survival. In a Perspective, Goulson and Nicholls discuss the anthropogenic effects on bee foraging, including the use of neurotoxic pesticides, diverse influences of urbanization, and emerging concerns such as electromagnetic radiation and particulate pollution. Combinations of these stressors may synergistically affect bee behavior, affecting their ability to thrive. Although mitigations in urban spaces have shown some improvements in bee foraging, mitigations in farmland pose a much greater challenge. —GKA Science, abn0185, this issue p. 970

RESTORATION ECOLOGY

Microbes repairing degraded soils Soils worldwide have become increasingly degraded by human activities, especially in drylands. Land degradation negatively affects soil hydrological functioning and thereby the ecosystem services that soil provides. Soil microbes may play an important part in the restoration of degraded soils, positively influencing moisture content and other physical features of soil. Coban et al. reviewed recent work on soil hydraulic properties, potential groups of microorganisms for hydrological soil restoration based on their resilience in dry soils, and future strategies for long-term restoration of degraded lands. —AMS Science, abe0725, this issue p. 990

Sci. Immunol. 7, eabi6112 (2022).

IMMUNOLOGY

A brake on lymphocyte activation Stimulation of antigen receptors leads to the formation of a protein complex called CBM on the scaffolding protein CARD11 and ultimately promotes T or B cell activation. The ability of CARD11 to recruit its binding partners to the CBM complex is regulated by multiple phosphorylation events. SCIENCE science.org

ANIMAL BEHAVIOR

Human impacts on bee foraging Bees are important pollinators that have evolved numerous learning, navigation, and communication abilities that allow them to forage for food. However, human activity can have a substantial influence on foraging by bees, affecting 4 MARCH 2022 • VOL 375 ISSUE 6584

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Soil microbiota as game-changers in restoration of degraded lands Oksana Coban*, Gerlinde B. De Deyn, Martine van der Ploeg

BACKGROUND: Soil, the living skin of Earth, provides ecosystem services critical for life: Soil acts as a filter and store of water, provides a growing medium that supplies plants and heterotrophs with water and nutrients, offers habitat for a large diversity of organisms, and is the source of most of our antibiotics. Humanity is increasingly challenged by the combination of climate change, population growth, and land degradation, including carbon loss, biodiversity decline, and erosion. In particular, land degradation reduces soil hydrological functioning and thereby several other ecosystem services. Such impacts occur through alterations of hydraulic functioning, infiltration and soil moisture storage, carbon cycling, biological activity, transport of nutrients and contaminants, and plant growth. Impacts of global environmental change and associated soil degradation need to be understood and reversed as biodiversity, food production, climate regulation, and people’s livelihoods are increasingly affected by soil

ecosystem degradation. The interplay between soil biota and soil hydrological functioning plays an essential role in many biogeochemical cycles, including the water and carbon cycles. Microorganisms dominate soil life and perform an array of vital soil functions by regulating nutrient cycling, decomposing organic matter, defining soil structure, suppressing plant diseases, and supporting plant productivity. The presence of microorganisms and their activity can affect soil structure and hydraulic properties in multiple ways. Case studies indicate the potential of microorganisms as game-changers toward the restoration of soil functioning. However, the role of soil microbiota in forming and sustaining soils has historically been overlooked.

decade, there have been an increasing number of studies suggesting the use of microorganisms as ecosystem mediators, particularly to enhance crop production and to engineer microorganisms for dryland restoration. Most current experimental approaches focus on monitoring changes in the microbial community that can be correlated with land restoration; however, microorganisms are also facilitators of ecosystem change, not just followers. We review how microorganisms can help address different types of land degradation, with a focus on physical soil loss and transformation, loss of soil chemical properties, and contamination. We discuss potentially the most valuable groups of microorganisms for soil restoration (namely, plant growth– promoting rhizobacteria, nitrogen-fixing bacteria, and mycorrhizal fungi), emphasizing drylands and advances in plant-microbe interaction studies. We review known effects of microorganisms on soil physical and, specifically, hydraulic properties at pore scale and discuss future strategies for the longterm restoration of degraded lands. We also identify the methodological challenges that have so far hampered progress in understanding soil biophysical processes. OUTLOOK: Microorganisms can play the lead-

ADVANCES: It has been proposed that micro-

bial communities not only are an indicator of ecosystem health and restoration level but also can be manipulated to enhance the recovery of degraded ecosystems. In the past

ing role in restoring degraded lands, improving soil hydraulic properties such as infiltration and water retention and reducing soil hydrophobicity, which together can facilitate ecosystem restoration. We advocate for research on mechanisms to restore degraded soils with the use of microorganisms. Given the critical role of freshwater availability to terrestrial life and the paucity of studies on hydrological restoration, we especially advocate for research on the hydrological restoration of degraded soil using microorganisms. We propose that microorganisms can improve soil hydraulic properties such as infiltration and water retention and reduce soil hydrophobicity. Along with new organic matter derived from microbes, this will promote plant growth and facilitate further ecosystem restoration. Such a restoration strategy requires collaboration across the research fields of microbiology and soil hydrology, of which there has been very little to date. Understanding the dynamics of soil microbes and connected hydrological processes would create the foundation for restoration practices that can return resilience to the soil ecosystem. Department of Environmental Sciences, Wageningen University & Research, Wageningen, Netherlands. *Corresponding author. Email: [email protected] Cite this article as O. Coban et al., Science 375, eabe0725 (2022). DOI: 10.1126/science.abe0725

Degraded land in Libya. In degraded lands, a decrease in soil nutrients and organic matter, deterioration of soil structure, increase in salinity, water deficiency, and physical instability can be observed. 990

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RESTORATION ECOLOGY

Soil microbiota as game-changers in restoration of degraded lands Oksana Coban*, Gerlinde B. De Deyn, Martine van der Ploeg Land degradation reduces soil functioning and, consequently, the services that soil provides. Soil hydrological functions are critical to combat soil degradation and promote soil restoration. Soil microorganisms affect soil hydrology, but the role of soil microbiota in forming and sustaining soil is not well explored. Case studies indicate the potential of soil microorganisms as game-changers in restoring soil functions. We review the state of the art of microorganism use in land restoration technology, the groups of microorganisms with the greatest potential for soil restoration, knowledge of the effect of microorganisms on soil physical properties, and proposed strategies for the long-term restoration of degraded lands. We also emphasize the need to advance the emerging research field of biophysical landscape interactions to support soil-plant ecosystem restoration practices.

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oil, the living skin of Earth, provides ecosystem services critical for life: It acts as a filter and store of water, provides a growing medium that supplies plants and heterotrophs with water and nutrients, offers habitat for a large diversity of organisms, and is the source of most antibiotics (1). Many of these services are created by soil life, which interacts with the complex biological, chemical, and physical dimensions of soil. When soil life disappears or degrades as a result of environmental disturbances, ecosystem services are also affected. The combination of climate change, human population growth, and soil degradation—including carbon loss, biodiversity decline, pollution, and erosion— represents an increasing challenge to humanity (2). At present, one-third of all global land surfaces are degraded to some extent (3), and 24 billion metric tons of fertile soil are lost every year (4). It is estimated that 50 million people may be displaced in the next decade as a consequence (4). Therefore, urgent efforts are needed to find solutions to restore wellfunctioning living topsoil in the coming years. Soil water represents only 0.05% of the global freshwater stocks, yet it is essential in supporting terrestrial life. The crucial role of soil moisture in the Earth system and in global environmental change is well studied (5, 6). Global environmental change and unsustainable land management can irreversibly reduce soil moisture retention properties (7, 8) and so can affect all life that depends on soil moisture. Moreover, short and extreme weather fluctuations can be as influential as gradual climate change in driving change in ecosystems (9). In addition, land degradation has a

Department of Environmental Sciences, Wageningen University & Research, Wageningen, Netherlands. *Corresponding author. Email: [email protected]

Coban et al., Science 375, eabe0725 (2022)

negative impact on soil hydrological functions. Soil hydraulic functions include rainfall infiltration and storage of soil moisture, which in return provide water to the soil-plantmicrobial and faunal ecosystem components. The interplay between soil biology and soil hydrological functioning performs an essential role in the hydrological cycle: Biophysical interactions regulate the water flux to the atmosphere through evapotranspiration, while the influx is dependent on adequate water supply from the atmosphere to the soil. Biophysical landscape interactions are those biotic and abiotic processes that influence the evolution of the landscape over time (10). Those interactions influence soil properties, which can lead to a shift in soil moisture regime attributable to, for example, the formation of soil crusts (8). Such regime shifts are different from common hysteresis effects associated with repeated wetting and drying of the soil (11); they are associated with an actual physical change of soil pore connectivity and structure (8). Changes in land management or climate can lead to a sudden shift in an ecosystem from one stable state to another (8, 9, 12, 13). The concept of alternative stable states was proposed as a way to understand system behavior in ecology (14) and physics (15). The transition between states is a consequence of nonlinear feedbacks initiated by “disruptive” perturbation (16)—for example, from a healthy ecosystem to a collapsed one, or from one type of ecosystem to another. For soil ecosystems, evidence for the occurrence of alternative stable states is scarce (7), but it can be expected that soil-degrading processes such as intense droughts, erosion, and landslides can trigger or at least contribute to state shifts and collapse. The actual mechanism of such changes would likely include drought-induced posi-

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tive feedbacks among erosion, loss of organic matter, and reduction of soil water retention (7, 17). Whether such shifts lead to irreversible changes in soil moisture availability remains debated; for some ecosystems, these altered trajectories may be reversible on human time scales. A healthy soil ecosystem sustains itself, biological productivity, and environmental quality within a landscape and promotes plant and soil biota health (18). These coupled components of the soil ecosystem are affected by land use and landscape position. In turn, differences in microbehavior in the soil ecosystem driven by the interplay of heterogeneous soil and flora and fauna dynamics affect emergent soil characteristics locally and across the landscape and determine soil resilience. The role of microbiota in forming and sustaining landscapes has been historically overlooked. In recent years, new field observations have given rise to the concept of “biophysical landscape interactions,” which implies that the physical mechanisms of soil functioning should be reevaluated to include biotic processes (8). Microorganisms dominate soil life and perform an array of vital soil functions by regulating nutrient cycling, decomposing organic matter, suppressing soil-borne plant diseases, defining soil structure, and supporting plant productivity. Hence, soil microorganisms are facilitators of soil ecosystem change. On the other hand, microbial community structure and diversity can also be used as indicators of soil health (19), and research so far has mainly focused on how microbial communities respond to environmental change (20). In an opinion paper on restoration ecology published 13 years ago, it was proposed that microbial communities are not just indicators of change but also underpin the recovery of degraded ecosystems when managed well to promote the restoration process and system health (21). Since then, an increasing number of studies have explored how microorganisms can be used as ecosystem mediators (22), particularly to enhance crop production (23, 24) and to engineer dryland restoration (25). These studies indicate the potential of microorganisms to be game-changers in restoring soil functions. To move this new research field of microbial soil restoration forward, we discuss a number of factors that need to be considered and describe the state of the art in the field. Furthermore, we discuss the groups of microorganisms that have potential in land restoration along with their effects on soil properties. We explore hydrological restoration from local to landscape scale and identify how soil microbes and microbe-plant interactions improve and sustain soil hydrological functioning and thus are necessary to ecosystem resilience and health. 1 of 10

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Groups of microorganisms used in restoring degraded lands

Restoration of degraded ecosystems can be slow and unsuccessful if conditions are unfavorable for the development of soil biota (26). Because soil biota is an important driver of plant community development (27, 28), the successful restoration of terrestrial ecosystems may depend on the manipulation of soil organisms (21). With respect to their impact on plant growth, nutrient cycling, and soil structure, three main groups of beneficial microorganisms can be distinguished: plant growth–promoting rhizobacteria (PGPR), nitrogen-fixing bacteria, and arbuscular mycorrhizal fungi (AMF) together with ectomycorrhizal fungi (EMF). As a possible fourth group, cyanobacteria in biological soil crusts (BSCs) are able to increase soil nutrient availability by fixing nitrogen and improving soil structure at the surface (29, 30). Examples of beneficial microorganisms as remediation agents are shown in Fig. 1. PGPR are loosely defined as soil bacteria that colonize the plant roots and enhance plant growth. Enhancement can occur in a direct or indirect way, and only some of the mechanisms involved have been characterized so far (31). Among direct mechanisms are nitrogen fixation, solubilization of minerals (e.g., phosphorus) and enhancement of their uptake by plants, synthesis of phytohormones, and chelation of metals to make them available to plant roots. The addition of PGPR has also been shown to increase crop yield by protecting plants from pests, parasites, or diseases (32). Agricultural yields are often limited by N availability, and the natural N input into the biosphere is mostly provided by biological N fixation by bacteria and archaea. Nitrogenfixing organisms or diazotrophs can be freeliving or exist in a symbiotic relationship with their host plant (33). Among different forms

of association, symbioses in which rhizobacteria live within root nodules are the most efficient in terms of making N available to the plant. Root nodule symbioses between leguminous crops and rhizobia result in particularly high rates of N fixation (50 to 465 kg N ha–1 year–1) and can occur in nearly all cropping systems (34). The world’s main cereal crops (rice, wheat, and maize) do not associate with rhizobia, and associative N fixation in cereals is often considered insignificant in comparison to nodulated symbiotic N-fixing plants (35). Manipulating bacteria to increase biological N fixation in nonleguminous plants has been a long-standing pursuit (36), but the expansion of symbiotic associative N fixation to other crops has been hindered by poor understanding of the genetic requirements that allow the host plant to associate with and benefit from diazotrophs (37). Technological advances during the past two decades, including nextgeneration sequencing, gene editing, and synthetic biology, enable genetic manipulation of plants and microbes at an unprecedented scale. However, it is expected that the development of nodulation in non-nodulating crops will still take a few decades (33, 38). EMF form symbiotic structures with the roots of woody plants and drive the nutrient cycle in forest ecosystems. In addition to nutrient supply, EMF have been shown to enhance water transfer between plants through their hyphal network, thereby increasing plant survival during drought periods (39). AMF form symbioses with the roots of ~80% of vascular plant species, helping them to acquire soil nutrients (especially phosphorus) in return for plant carbohydrates (40). In some cases, AMF provide most of the plant’s phosphorus needs, and all its phosphorus uptake may be of hyphal origin (41). Phosphorus is needed for all major metabolic processes (photosynthesis, respiration, energy transfer), and seed

Fig. 1. Land degradation types and examples of beneficial microorganisms acting as remediation agents. Coban et al., Science 375, eabe0725 (2022)

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formation and root strength depend on it. Associations between AMF and bacteria are important in the symbiosis with the plant roots because of the critical role they play in mycorrhizal functions (42, 43). Disentangling the very complex ecological roles of AMF requires a collaborative effort involving physiologists, molecular biologists, and ecologists. Recent advances in molecular genetics are helping to decipher the morphological, physiological, and genetic characteristics of AMF and to understand the molecular mechanisms of symbiosis among AMF, bacteria, and plants (44). Stable isotope probing (SIP), for example, makes it possible to study the interrelated effects of structure and function of the symbiont organisms (45, 46). Plant-microbe interactions

Plants in natural environments coexist with a wide variety of microbes (archaea, bacteria, fungi, viruses, and protists), collectively called the microbiome. The microbiome is vital to plant growth, productivity, and plant overall condition (47). The interactions can be pathogenic, commensal, or beneficial, depending on such factors as plant species, soil type, and environmental conditions (31). Plant-microbiome engineering aims to direct the interactions toward increasing crop yield. The understanding of plant-microbiome interactions remains rudimentary (31), but it has become clear that there is considerable scope for manipulation. In recent years, research into the application of microorganisms as biocontrol, biostimulant, and bioremediation agents has expanded (48–50). In agriculture, the primary research foci are product reliability and consistency (48). Better understanding of the mechanisms of plant-microbial interactions is key to increasing the benefits to be gained from their exploitation (51). Major developments in plant-microbe interactions have occurred in recent years thanks to advances in techniques for genomic studies and the increased availability of genomic information (52, 53). Such developments provide a basis for exploring plant-microbe interactions across chemical, molecular, metagenomic, and exometabolomic scales (54). For example, progress is being made toward large-scale microbial isolation and the synthetic production of communities (55). Next-generation sequencing techniques provide a necessary basis for the preparation of bacterial isolates that are further inoculated in germ-free plants. Systematic microbiota culture collections can be used in microbiota reconstitution experiments with germ-free plants to investigate the principles underlying community assembly and to identify microbiota-mediated functions important for plant health (56). Furthermore, novel methods of exometabolomics can help to unravel the mechanisms underlying 2 of 10

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the ability of plants to actively shape their microbiomes through insight into the molecular mechanisms of microbial-induced plantspecialized metabolism (57, 58). In field situations, the microbiota present in the soil acts as the main pool from which plants can select their rhizosphere or root microbiome. Hence, soil management and its impact on the soil microbiome remain of critical importance for engineering root microbiota. On the other hand, the agricultural environment and the genotypes of the cultivated plants have an impact on individual microbial genotypes (59). This suggests that manipulation of plants and cropping systems is the key to sustainable biocontrol. When the biology underpinning plant-microbe interactions is well understood, inoculation with individual bacterial isolates can also be effective (60). Further protection for plants is provided by the initial soil microbiome acting against pathogen invasion or by promoting plant resistance and crop productivity (61). This provides a basis for engineering root microbiota and for greater understanding of the functions of root and soil microbiota in plant growth and health. Choice of microorganisms for dryland restoration

Many studies have focused on the resilience of microbial communities in dry soils and on the effects of soil degradation on a microbial community structure (62, 63). These studies can help in selecting the most promising candidate species for degraded land restoration. Several species of algae have been shown to promote soil hydrological properties by breaking water repellency and improving water retention (64, 65). In studies of the response to desiccation, fungi showed higher resistance than bacteria to changes in water availability (63, 66), enabled by hyphae that may traverse air-filled soil pores to access nutrients and water. In bacterial communities, survival strategies for dry conditions have long been examined by testing physiological responses (67, 68). More recently, next-generation sequencing techniques have helped to further explore trends in abundances of major taxonomic groups as a function of soil moisture conditions (62, 63, 69). In the context of differences between arid and non-arid environments, or differential responses to desiccation stress, bacterial abundances vary in response to different moisture levels in three main ways: 1) With decreasing soil humidity, there is an increase in abundances of Cyanobacteria (69–71) and monoderm bacteria (i.e., prokaryotic cells surrounded by a single membrane) (68, 72); this latter group includes Actinobacteria (69, 73), Chloroflexi (62, 73), and Firmicutes (70, 73, 74). All of these bacterial taxa possess multiple mechanisms that help them withstand harsh environments including desiccation for extended periods, namely Coban et al., Science 375, eabe0725 (2022)

a thick peptidoglycan layer (75) and an ability to produce resting stages (e.g., endospores) (76). 2) Some of the terrestrial diderm bacteria [phyla such as Acidobacteria (62, 69) and Verrucomicrobia (62, 69)] follow the opposite pattern: Their abundances decline during dry-out and increase with rewetting. An exception is the class Chloracidobacteria of Acidobacteria that is distinct from this group (69, 77). 3) Other diderm phyla (e.g., Proteobacteria, Planctomycetes, Bacteroidetes) do not show a consistent response to aridity. Their abundances in dry soil have been reported to increase in some studies and to decrease in others (62, 69, 70, 74). Each of the three possible responses (increase, decline, no change) was also found in ribosomal synthesis of these phyla (63, 74), a response that usually serves as a proxy for potential activity, although rRNA concentration and growth rate are not always simply related (78). Different bacterial species can produce either hydrophilizing or hydrophobizing substances as a part of extracellular polymeric substances (EPSs) (79) and thereby affect soil hydrophobicity and water infiltration. Hydrophilizing properties were found for Bacillus sphaericus of the Firmicutes phylum (79). Furthermore, some Actinobacteria and Proteobacteria are able to degrade waxes and thereby reduce water repellency (80, 81). This indicates a promising potential use of these bacteria to improve soil wettability and break down hydrophobicity. All of these findings suggest the possible application in arid environments of either monoderm bacteria (Actinobacteria, Chloroflexi, Firmicutes) and Cyanobacteria or the generalists that expose drought-resistant patterns (e.g., fungi, algae, Proteobacteria). Recently, fungi have been shown to facilitate colonization of dry soil by bacteria (82), and therefore studies investigating co-inoculations of bacteria and fungi are also of interest. Types of land degradation and the state of the art of microbe use in land restoration

Land degradation is the loss of the intrinsic physical, chemical, or biological soil characteristics by natural or anthropogenic processes, resulting in a reduction or eradication of vital ecosystem functions (83). Land degradation estimations at the global scale are complex and vary between studies (Fig. 2). Because no formal classification of land degradation exists, we propose three principal and interconnected processes of degradation (84): 1) The physical loss or physical transformation of soils (e.g., associated with erosion, landslides, drought, severe fires, soil compaction, and sealing); 2) The loss of soil chemical properties, such as decline in fertility and organic carbon content

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due to acidification, salinization, or nutrient deficiency; and 3) Soil contamination. Microorganisms can promote soil restoration in each of these cases. Here, we discuss each of the three degradation types with special emphasis on driving causes most commonly associated with global environmental change, namely drought, fire, and soil salinization (Fig. 3). Physical loss of soil

Physical land degradation can affect soil at all latitudes and on all continents. One driving cause is the increased frequency and severity of drought. Drylands already cover an estimated one-third of Earth’s terrestrial surface (85) and are expected to increase in size as a result of climate change and human activities (86). In drylands, strong winds and ultraviolet radiation lead to decreases in soil nutrients and organic matter, deterioration of soil structure, and increases in salinity, water deficiency, and physical instability (87). Under such stresses, plants suffer from a number of biochemical, morphological, and physiological modifications that suppress their growth and productivity (88). PGPR found in dry environments are well adapted to extreme environmental conditions such as drought, heat, and high salinity, providing essential nutrients from soil or through N fixation and enhancing plant tolerance to abiotic and biotic stresses (89). PGPR in drylands possess unique traits, including a stronger expression of genes related to dormancy and osmoregulation as well as a weaker expression of genes regulating nutrient cycling and catabolism (89). They are also capable of increasing the soluble sugars content and chlorophyll content in the leaves, which enable drought stress tolerance in plants (90). Highly desiccation-tolerant PGPR, such as Actinobacteria, produce the sugar trehalose, which increases their abiotic stress tolerance and at the same time symbiotically protects plants against drought by induction of their stressresponse genes (91). Over the past decade, there has been increased interest in the development of biofertilizers based on desert PGPR that can promote plant tolerance against abiotic stress in drylands (88, 89). Inoculating soil in degraded drylands with biological crusts can contribute to the recovery of ecosystem functions at the landscape scale, such as erosion resistance by improving soil stability and nutrient cycling. Using living inoculum has shown to be more effective in drylands than replacing topsoil or organic carbon additions (92). Low-severity fires, such as grassland fires or prescribed fires for landscape management, enhance soil properties by reducing surface flow and erosion and increasing soil organic matter and nutrient content (93). In contrast, wildfires greatly change soil physical properties 3 of 10

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Fig. 2. Development of land degradation estimations. Top left: Total estimated land degradation. Top right: Estimated land degradation per continent. These graphs are based on data from Gibbs and Salmon (2015) and IPBES (2018) (177, 178); Antarctica is excluded in both panels. Variations between these estimations have not led to a conclusive estimate of spatial coverage and severity of land degradation at the global scale (179). For this reason, IPBES (2018) uses a convergenceof-evidence approach based on global change issues.

Fig. 3. Main types of land degradation and main types of useful bacteria.

(altered pore size, water repellency, stability of aggregates), biological composition (vegetation loss, changes in microbial communities), and chemical properties (increase in organic carbon due to combustion, volatilization of nitrogen and phosphorus), with detrimental effects (94, 95). Burned soil may become hydrophobic as a result of accumulation of hydrophobic substances on the soil surface after burning of organic materials (96). Moreover, the exposed soil is much more vulnerable to wind (97) and water (96) erosion during the first few years Coban et al., Science 375, eabe0725 (2022)

following wildfires. Loss of topsoil due to erosion is the primary risk of failure for forest replanting. Another consequence of the wildfires is the formation of hazardous organic compounds such as polycyclic aromatic hydrocarbons (PAHs) as a result of combustion processes of the plant biomass (98). Soil microorganisms play an important role in soil function recovery after fire. PGPR have been shown to enhance nutrient availability and promote plant growth in fire-impacted soil (99, 100). The inoculation of soil with ectomycorrhizal fungi in burned forest also stimulates plant growth (101), making them attractive for assisted forest regeneration in fire-damaged soils. Alternatively, the growth of locally indigenous soil microorganisms can be stimulated with organic matter, oxygen, specific plant species, or other stimulating factors through so-called biostimulation (102). Biostimulation enhances the natural biodegradation potential of the indigenous microbial community and has been shown to be a most promising strategy for PAH removal from burned forest soil (103) and from contaminated soil (104). To combat hydrophobicity of soils, wax-degrading Actinobacteria have successfully been used to reduce water repellency, either via Actinobacteria inoculation in soil (105) or when still present in soil via their biostimulation (106), which is a more practical and economical approach.

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Loss of soil fertility

Since the Green Revolution in the 1950s and 1960s, agricultural production has increased worldwide with the aim of feeding an increasing world population. Global food demand will rise by another 70% in the coming decades, emphasizing the need for intensive crop production (107). The intensive cultivation of crops can lead to the exhaustion of soil organic matter and soil nutrient reserves, resulting in soil degradation (108). Driven by increased understanding of the role of microorganisms in soil health and crop productivity, the use of agronomic practices to restore soil fertility through organic matter addition, microbial N fixation, and nutrient recycling (i.e., regenerative agriculture) is also increasing rapidly. Such practices include no-tillage, rotating crops and growing cover crops, returning crop residue into the soil, using biofertilizers or biopesticides, and applying organic fertilizers such as manure, biochar, and compost (109). It has been shown that biofertilizers (i.e., inocula of fungi and bacteria) can be combined with organic fertilizers to reduce the need for chemical fertilizers and to use soil resources more efficiently (110). As for the biopesticides, these are less toxic than abiotic pesticides, reducing environmental pollution, and can be more effective than chemical pesticides in the long term (110). Soil organic matter, biopesticides, and biofertilizers provide 4 of 10

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a foundation to achieve food security for the growing population and restore soil fertility (48). Indeed, next to soil chemical and soil physical parameters, microbial soil indicators have been used as a proxy to measure soil fertility and soil health levels more generally (19). Remediation of saline soils

Each year, around 1 to 2% of fertile soils are being degraded worldwide as a consequence of salinity (111), and about 20% of irrigated land is currently already affected by salinization (112). Soil salinity can be due to cations such as sodium (Na+) and anions such as chloride (Cl–), among others. Exposure to salinity increases the concentrations of Na+ and Cl– ions and decreases the availability of other ions such as potassium (K+) to plants. Sodium ions not only cause plant cell injury but also degrade soil structure. By contrast, K+ is essential to plant cells as a major inorganic nutrient and an osmotic regulator (113). Along with ionic imbalance, long-term salinity reduces the ability of plants to extract water from the soil, making the salt and drought problems of plants closely related (113). Salinity occurs in agricultural soils in the arid and semi-arid regions of the world, but also in some coastal regions (e.g., the Netherlands) as a result of rising seawater level. Consequently, interest in salinity-tolerant plants and sustainable methods for the bioremediation of saline soils is also growing (111). Biological remediation by the application of salt-tolerant, or halophilic, PGPR and AMF is a sustainable alternative to conventional physical and chemical treatments of saline soil (111, 114, 115). Halophilic PGPR can remediate saline soils directly via improving nutrient status, soil structure, organic matter, pH, electrical conductivity, and deposition of ionic salts in soil (116). These PGPR are increasingly seen as an efficient tool to mitigate salinity stress in plants through mechanisms stimulating multidirectional physiological, biochemical, and molecular responses. In particular, inoculation with halophilic PGPR and AMF increases K+/Na+ ratios, which are beneficial in maintaining ionic homeostasis in the cytoplasm or Na+ efflux from plants (114, 115). Lately, the application of halophilic PGPRs from the rhizosphere of halophilic plant species has been actively explored as a means to stimulate plant growth and increase the salt tolerance of nonhalophytic crops (111, 117). Recent studies on the underlying mechanisms have shown that halophilic PGPR can modify the expression in plants of several genes responsible for the amelioration of salinity stress (118, 119). The connection among plant stress responses, signaling molecules, and microbiome assembly can be used to modify the phytomicrobiome for the benefit of stressed Coban et al., Science 375, eabe0725 (2022)

plants and can be explored further for the development of stress-resilient “smart agriculture” (120–122). Soil contamination

Soil can become contaminated by chemicals originating from various sources, including agricultural and industrial activities, dumping waste, and urbanization. Soil pollution can produce negative agricultural, industrial, urban, and environmental effects such as reduced soil fertility, water pollution, reduced plant growth, and modified soil biodiversity. These can cause subsequent soil erosion and degradation, ultimately changing the whole ecosystem (123). Information about the global extent of soil pollution is lacking, as only some countries undertake national surveys of soil pollution (124). However, the information that is available is cause for concern. For example, China has categorized 16% of all its soils as polluted (125). Among physical, chemical, and biological approaches for remediation, interest in the latter has amplified in recent years as societies turn to sustainable and green solutions to solve environmental problems (126). Bioremediation of contaminated soils refers to the degradation of pollutants through microbial metabolic activities by mostly indigenous microorganisms (102). PGPR also have an indirect positive impact on removal of contaminants by plants (phytoremediation)—for example, by stimulating plant growth and increasing contaminant bioavailability—and this role can be manipulated to improve the efficacy of phytoremediation (127). Microbe-assisted phytoremediation has shown to be efficient in restoring sites contaminated by heavy metals, pesticides, and hydrocarbons (128). This biotechnology can kick-start further recovery of degraded ecosystems, leading to much faster biodiversity restoration (128). For example, inoculation of contaminated soil with fungi when reintroducing vegetation not only enhances the extraction of heavy metals from the polluted soils but also enables the plants to establish themselves on degraded soil and thereby improve soil quality and health (129, 130). The effects of remediation on the restoration of biodiversity remains to be explored further. For example, one recent study considers how the composition and structure of fungi after phytoremediation are driven by the plant species present (131). A wide variety of microorganisms and metabolites that can degrade pollutants are still largely unexplored, and better understanding of their metabolism will enable their use in bioremediation. Recently, the potential of extremophiles that survive in environments with high concentrations of metals, radionuclides, or other pollutants has been explored (132, 133). Next-generation sequencing approaches can help to discover microbial diversity and metabolic functions that

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predict the presence and extent of contamination, characterize the process of natural attenuation by unculturable microbes, and clarify the impact of biostimulation on microbial communities (134). Effects of microorganisms on soil physical properties

Microbes affect soil structure by direct and indirect processes, including (i) moving primary particles along cell or hyphal surfaces; (ii) adhering particles together by their secretions, such as EPSs; (iii) enmeshing and binding aggregates by fungal hyphae and actinomycete filaments; and (iv) producing hydrophobic compounds that coat pore walls, particularly in fungi. A major factor in soil structure formation and stabilization is the soil organic matter, which increases soil aggregation. Microbes, including their remains or necromass, can make up more than half of soil organic carbon (135, 136), highlighting the role of microbes along with macroorganisms and plants in strongly related soil physical properties, such as porosity and aggregate stability. Soil aggregates support root growth, resistance to erosion, carbon storage, and waterholding capacity (137), and a well-aggregated soil structure is fundamental to ensure healthy functioning of soil (43). Bacteria and fungi excrete mucilages that play important roles in soil microaggregate stabilization by gluing soil particles together (110, 137). They also take part in macroaggregate stabilization by entangling particles within the hyphae network and through production of EPSs (110, 137). Also, AMF are known to produce glomalin-like proteins that play an important role in soil stabilization, carbon storage, and soil aggregate stability and thereby decrease soil susceptibility to erosion (138). Although the role of bacteria and fungi in soil aggregate formation and stability is established, experimental studies on the application of microorganisms for soil structure improvement are scarce (139, 140). Effects of microorganisms on soil hydraulic properties

Aggregate stability, soil structure, organic matter, and microtopography are all attributes associated with soil hydraulic properties. Microorganisms affect soil hydraulic properties at the pore scale in multiple ways (Fig. 4): by biofilm formation, EPS excretion, exudation of binding agents (e.g., glomalin-like proteins), particle enmeshment by fungi, production of water-repellent compounds, and alteration of soil surface microtopography. Biofilms are aggregations of microorganisms whose cells adhere to each other or to a surface, often embedded within a self-produced matrix of EPS (141). Biofilms can form as a response to an environmental stress, enabling 5 of 10

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Fig. 4. Comparison of healthy and degraded land. Left: Hydrological fluxes in healthy soil. Right: Possible changes in soil hydrological fluxes as a result of soil degradation. Microbial processes are circled. Degraded land is experiencing a decline in microbial diversity as well as reduced amounts of extracellular polymeric substances (EPSs) and soil organic matter (SOM) that lead to a decrease in water-holding capacity and other hydrological changes, as shown here.

the cells to remain in a favorable niche and form mutualistic relationships (141). The key mechanism for changing soil hydraulic properties with biofilms is bioclogging, which is one of two main applications of a new branch of geotechnical engineering called microbial geotechnology (142, 143). It aims for a manipulated partial clogging of porous media by microorganisms and their by-products, in order to reduce soil porosity and hydraulic conductivity. Another application, biocementation, uses microorganisms to improve the engineering properties of soils and is beneficial for increasing soil strength and stiffness. Bacterial EPS formation can improve soil water retention and reduce desiccation (144Ð146). EPS improves soil water-holding capacity in three different ways. First, certain polysaccharides (e.g., xanthan, dextran, scleroglucan) are a component of EPS and are known for their hydrophilic properties. By formation of clay- or sand-polysaccharide associations with soil microorganisms, a general increase in waterholding capacity may be observed (146, 147). In particular, a creation of hydrated microenvironments via production of alginate as an important component of EPS that decrease desiccation stress was shown for Pseudomonas (148). Second, EPS promotes the formation of soil aggregates and thereby soil water retention as the small intra-aggregate spaces hold water firmly (149, 150). Third, EPS modifies soil surface water repellency, causing an increase in the number of hydrophobic micropores that inhibit water evaporation (149, 151, 152). Coban et al., Science 375, eabe0725 (2022)

A better understanding of the role of biofilms in soil hydraulic properties requires more studies, following a standardized methodology (Box 1). Microorganisms are also able to produce substances other than EPSs to protect them in dry conditions. In bacteria, such compounds include disaccharide trehalose, which is responsible for osmotic stress response, and a polysaccharide a-glycan that increases desiccation tolerance in bacteria (91, 153). Microbial activity affects soil micro- and macroporosity via the exudation of binding agents and particle enmeshment by fungi that promote aggregate formation (154). Glomalin-related soil proteins, soil particle gluing agents that are produced by AMF, also improve soil moisture retention properties by increasing aggregate stability (155, 156). Soil water repellency or hydrophobicity occurs when hydrophobic organic compounds accumulate at the soil surface. Those compounds can be derived from plant leaves and roots as well as from microorganisms. Examples of hydrophobic microbe-derived compounds are ergosterol and glomalin-related soil proteins (157). Fungal hyphae and bacterial excreta promote aggregate stability but at the same time can increase soil water repellency (158). On the other hand, some bacterial species have hydrophilizing properties, or have ability to degrade waxes, and thereby reduce hydrophobicity (80, 81). BSCs in arid and semi-arid environments develop when communities of extremely drought-

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resistant microorganisms dominated by cyanobacteria colonize the soil surface (159). They influence soil hydrology because they alter soil properties. Although there is abundant literature on the effects of BSCs on soil water repellency and infiltration, reports are contradictory. Some authors observed lower infiltration rates and higher runoff for soils with BSCs, relative to soils with low levels or no microbial cover (159, 160); others report positive effects (161) or no effects at all (162). These discrepancies can be attributed to methodological approaches, rainfall characteristics, soil factors, and/or natural variations of the BSC composition on crust functioning (160, 163). Because infiltration rates are controlled by soil surface structure, the rough surface microtopography of BSCs, characteristic for semiarid cool and cold drylands, increases water infiltration and reduces runoff. On the other hand, BSCs in hyper-arid warm regions tend to be smooth and flat because of the absence of frost heaving, thereby reducing water infiltration and increasing runoff. Mixed effects are observed in arid regions (164, 165). Conclusions and future prospects

Ecosystems vary greatly in the degree to which they are able to recover naturally from human disturbance. Conservation efforts have increasingly focused on active or natural restoration of degraded ecosystems in order to restore ecosystem services and biodiversity (166). In some cases, only minor interventions such as removing human disturbances (e.g., fire, 6 of 10

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Box 1. Methodological challenges. Although a number of studies have explored the role of biofilms in soil physical processes, most of these studies face methodological issues. Methodological needs are summarized below. Setting proper scientific controls In studies (142, 180, 181) investigating effects of a microorganism on soil hydraulic properties, the control group did not include all of the constituents of the media that had been used along with the microorganism in the treatment group. Considering that soil additives have been shown to affect soil hydrology (182), a lack of the proper negative control prohibits distinguishing effects of the tested microorganism from the effects of media. Combining microbiological and hydrological methodologies For measurements of soil hydrophysical properties that may take weeks, the laboratory needs to be temperature- and humidity-controlled. Low temperature is important for minimizing microbial growth that could alter hydraulic parameters while these measurements take place. Although some studies use elevated temperature settings for stimulation of evaporation in order to reduce the required duration of the experiment (180), use of lower-humidity settings is recommended over increasing temperature in such experiments (181). Standardizing the hydrological methodology Standardization of methodological procedures is vital for cross-comparison of data from different experiments, consistency between replicates, and minimizing risk of procedural bias. Soil compaction has been shown to affect bacterial abundances in inoculated soil (142) and also strongly affects soil hydraulic properties via changes of bulk density (183). For soil sterilization, double sterilization or gamma-radiation appear to be most efficient (184, 185), although their alteration of soil chemical and physical properties should be considered (186). Also, a temperature dependence of water retention curves for various types of soils has been shown (187), and comparisons between different studies would be facilitated by the use of low temperature (as explained above). Monitoring of microbial growth Hydrological methodologies require a substantial amount of time (e.g., several days or even weeks), and microbial growth should be evaluated and reported. A study (181) showed that carbon deficit assessment demonstrated more consistent results than cell counting, a method used in most similar experiments (64, 142); therefore, careful consideration should be given to the choice of monitoring method. How microbial abundance affects soil hydraulic properties remains to be tested. Microorganism addition to a sterilized, not living soil “Proof of concept” studies evaluating the effect of a single microorganism in sterilized soil are useful for early investigational stages (65, 180, 181). The next step for such studies should be focused on approaches to enrich a microorganism in field soil and investigation of the effects of such enrichment on the entire microbial community. Finding suitable microorganism(s) Reports on enhancement of soil hydraulic properties after incubation with microorganisms are scarce in the current literature. Effects on soil hydraulic properties of only a few bacterial species (142, 180, 181), fungi (188), and algae (64, 65) have been reported. In addition to these, several studies on arid and semi-arid land restoration using biological crusts are available (71, 161, 189). We also suggest exploring the synergistic effects of consortia of microorganisms that can provide additional benefits for improving soil quality by means of, for example, N fixation and soil structure formation.

grazing) are sufficient to facilitate unassisted recovery. For severely degraded ecosystems, in particular when soil hydrology is affected (167), greater efforts may be needed to accelerate and influence the successional trajectory of recovery. Given the spread of land use and change by humans globally, we need approaches that return healthy soil ecosystems in more timely and steady trajectories than are achieved with the methods that are currently Coban et al., Science 375, eabe0725 (2022)

available (168). In agricultural systems, land use and management strategies critically affect the rate and trajectory of recovery (168). Agricultural practices such as reduced tillage, crop rotation, organic amendments, and reduced fertilizer use where there was overuse can help prevent disruption of beneficial plant root symbioses, such as with N-fixing bacteria and mycorrhizal fungi, and entire microbial communities.

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Although beneficial soil-borne organisms are already naturally present, the aim in soil restoration is to enhance their abundance and activity by either inoculation or enhancement of their growth. Direct injections of bacteria together with growth substrates into the soil has disadvantages, such as clogging of the soil pore spaces and higher concentrations of organisms and substrates near the injection points (169). Furthermore, a single microbial species is not likely to adapt and survive in all soil environments (170) and thus geographical differences in microbial soil composition should be considered (171). Furthermore, commercially available PGPR may affect the native microbial population, unlike indigenous bacteria that are well adapted to the local environment (100). Instead, a strategy more likely to be successful is to introduce microorganisms as a part of a healthy soil inoculum as an aid to plant community reestablishment, especially in conditions where the topsoil has been replaced (26, 110, 172). Another way to stimulate the establishment of healthy soil microbiomes in degraded soils is through amendments with (noncontaminated) sewage sludge (173) or another organic substrate that matches the needs of the degraded soil. Given the risks to human health from pathogenic microbes of such organic materials as manure and sewage biosolids (174), alternative approaches to enhance soil quality are needed. Novel amendments are currently being developed that are free of such risks and are still able to enhance soil hydraulic properties, while at the same time stimulating microbial growth and increasing soil organic matter content (175). The longterm effects of these amendments on soil properties and on entire microbial communities remain to be studied. Before upscaling such approaches, detailed and systematic studies are needed in which a particular microorganism is enriched in living soil and its long-term effects on soil properties and soil microbial communities are tracked; such studies are entirely lacking at present. Given the critical role of freshwater availability in terrestrial life and the paucity of studies on hydrological restoration, we especially advocate for research on the hydrological restoration of degraded soil using microorganisms. We propose that microorganisms can improve soil hydraulic properties such as water infiltration and water retention and reduce soil hydrophobicity. Along with new organic matter derived from microbes, this will promote plant growth and facilitate further ecosystem restoration. Such a restoration strategy requires collaboration across the research fields of soil microbiology and soil hydrology, of which there has been very little to date (176). Understanding the interaction between soil microbial and hydrological dynamics will create the 7 of 10

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foundation for restoration practices that can return resilience to the soil-plant ecosystem.

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O.C. acknowledges support from the NWO Open Mind grant, G.B.D.D. thanks FoodShot Global for their support through the GroundBreaker Prize, and all authors thank A. Van Dijk for valuable comments and English proofreading. Author contributions: Conceptualization: O.C., M.v.d.P., G.B.D.D. Visualization: O.C., M.v.d.P. Funding acquisition: M.v.d.P., G.B.D.D. Writing–original draft: O.C., M.v.d.P., G.B.D.D. Writing–review and editing: O.C., M.v.d.P., G.B.D.D. Competing interests: The authors declare that they have no competing interests.

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crowd annotation to assign transcriptomic cell types with the best knowledge available.

GENETICS

RESULTS: We sequenced 570,000 cells using

Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly

droplet-based 10x Genomics from 15 dissected tissues as well as whole heads and bodies, separately in females and males. We also sequenced 10,000 cells from dissected tissues using the plate-based Smart-seq2 platform, providing deeper coverage per cell. We developed reproducible analysis pipelines using NextFlow and implemented a distributed cell-type annotation system with controlled vocabularies in SCope. Crowd-based annotations of transcriptomes from dissected tissues identified 17 main cell categories and 251 detailed cell types linked to FlyBase ontologies. Many of these cell types are characterized for the first time, either because they emerged only after increasing cell coverage or because they reside in tissues that had not been previously subjected to scRNA-seq. The excellent correspondence of transcriptomic clusters from whole body and dissected tissues allowed us to transfer annotations and identify a few cuticular cell types not detected in individual tissues. Cross-tissue analysis revealed location-specific subdivisions of muscle cells and heterogeneity within blood cells. We then determined cell type–specific marker genes and transcription factors with different specificity levels, enabling the construction of gene regulatory networks. Finally, we explored sexual dimorphism, finding a link between sex-biased expression and the presence of doublesex, and investigated tissue dynamics through trajectory analyses.

Hongjie Li†, Jasper Janssens†, Maxime De Waegeneer, Sai Saroja Kolluru, Kristofer Davie, Vincent Gardeux, Wouter Saelens, Fabrice P. A. David, Maria Brbic´, Katina Spanier, Jure Leskovec, Colleen N. McLaughlin, Qijing Xie, Robert C. Jones, Katja Brueckner, Jiwon Shim, Sudhir Gopal Tattikota, Frank Schnorrer, Katja Rust, Todd G. Nystul, Zita Carvalho-Santos, Carlos Ribeiro, Soumitra Pal, Sharvani Mahadevaraju, Teresa M. Przytycka, Aaron M. Allen, Stephen F. Goodwin, Cameron W. Berry, Margaret T. Fuller, Helen White-Cooper, Erika L. Matunis, Stephen DiNardo, Anthony Galenza, Lucy Erin OÕBrien, Julian A. T. Dow, FCA Consortium, Heinrich Jasper, Brian Oliver, Norbert Perrimon*, Bart Deplancke*, Stephen R. Quake*, Liqun Luo*, Stein Aerts*

INTRODUCTION: Drosophila melanogaster has

had a fruitful history in biological research because it has contributed to many key discoveries in genetics, development, and neurobiology. The fruit fly genome contains ~14,000 proteincoding genes, ~63% of which have human orthologs. Single-cell RNA-sequencing has recently been applied to multiple Drosophila tissues and developmental stages. However, these data have been generated by different laboratories on different genetic backgrounds with different dissociation protocols and sequencing platforms, which has hindered the systematic comparison of gene expression across cells and tissues. RATIONALE: We aimed to establish a cell atlas

for the entire adult Drosophila with the same genetic background, dissociation protocol, and

sequencing platform to (i) obtain a comprehensive categorization of cell types, (ii) integrate single-cell transcriptome data with existing knowledge about gene expression and cell types, (iii) systematically compare gene expression across the entire organism and between males and females, and (iv) identify cell type– specific markers across the entire organism. We chose single-nucleus RNA-sequencing (snRNAseq) to circumvent the difficulties of dissociating cells that are embedded in the cuticle (e.g., sensory neurons) or that are multinucleated (e.g., muscle cells). We took two complementary strategies: sequencing nuclei from dissected tissues to know the identity of the tissue source and sequencing nuclei from the entire head and body to ensure that all cells are sampled. Experts from 40 laboratories participated in

CONCLUSION: Our Fly Cell Atlas (FCA) constitutes a valuable resource for the Drosophila community as a reference for studies of gene function at single-cell resolution. All the FCA data are freely available for further analysis through multiple portals and can be downloaded for custom analyses using other singlecell tools. The ability to annotate cell types by sequencing the entire head and body will facilitate the use of Drosophila in the study of biological processes and in modeling human diseases at a whole-organism level with cell-type resolution. All data with annotations can be accessed from www.flycellatlas.org, which provides links to SCope, ASAP, and cellxgene portals.

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Tabula Drosophilae. In this single-cell atlas of the adult fruit fly, 580,000 cells were sequenced and >250 cell types were annotated. They are from 15 individually dissected sexed tissues as well as the entire head and body. All data are freely available for visualization and download, with featured analyses shown at the bottom right. SCIENCE science.org

The list of author affiliations is available in the full article online. *Corresponding author. Email: [email protected]. harvard.edu (N.P.); [email protected] (B.D.); steve@ quake-lab.org (S.R.Q.); [email protected] (L.L.); stein.aerts@ kuleuven.be (S.A.) These authors contributed equally to this work. Cite this article as H. Li et al., Science 375, eabk2432 (2022). DOI: 10.1126/science.abk2432

READ THE FULL ARTICLE AT https://doi.org/10.1126/science.abk2432 4 MARCH 2022 • VOL 375 ISSUE 6584

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GENETICS

Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly Hongjie Li1,2,3†, Jasper Janssens4,5†, Maxime De Waegeneer4,5, Sai Saroja Kolluru6,7,8, Kristofer Davie4, Vincent Gardeux9,10, Wouter Saelens9,10, Fabrice P. A. David9,10,11, Maria Brbic´12,8, Katina Spanier4,5, Jure Leskovec12,8, Colleen N. McLaughlin1, Qijing Xie1, Robert C. Jones6,7,8, Katja Brueckner13‡, Jiwon Shim14, Sudhir Gopal Tattikota15,16, Frank Schnorrer17, Katja Rust18,19, Todd G. Nystul19, Zita Carvalho-Santos20, Carlos Ribeiro20, Soumitra Pal21, Sharvani Mahadevaraju22, Teresa M. Przytycka21, Aaron M. Allen23, Stephen F. Goodwin23, Cameron W. Berry24, Margaret T. Fuller24, Helen White-Cooper25, Erika L. Matunis26, Stephen DiNardo27,28, Anthony Galenza29, Lucy Erin OÕBrien29, Julian A. T. Dow30, FCA Consortium§, Heinrich Jasper31, Brian Oliver22, Norbert Perrimon15,16*, Bart Deplancke9,10*, Stephen R. Quake6,7,8*, Liqun Luo1*, Stein Aerts4,5* For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell typeÐrelated gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

D

rosophila melanogaster has had a fruitful history in biological research, dating back to the experiments of Thomas Hunt Morgan more than a century ago (1), and has been at the basis of many key biological discoveries. The highly collaborative nature of the Drosophila community contributed to many of these successes and led to the development of essential research resources, including a high-quality genome (2), a large collection of genetic and molecular tools, and important databases such as Flybase (3), FlyMine (4), FlyLight (5), VirtualFlyBrain (6), and ModERN (7). The fly genome contains about 17,000 genes, including 13,968 protein-coding genes of which ~63% have human orthologs. Studies such as ModENCODE (8) and FlyAtlas (9) explored expression patterns in different tissues but lacked

cell-type resolution. Recent advances in singlecell technologies have enabled the transcriptomic profiling of thousands of cells at once, facilitating the creation of tissue-wide atlases. Several studies have already applied single-cell RNA sequencing (scRNA-seq) to multiple Drosophila tissues and developmental stages (10). However, these data were generated by different laboratories on different genetic backgrounds with different dissociation protocols and sequencing platforms, which has hindered the systematic comparison of gene expression across cells and tissues. Here, we present a single-cell transcriptomic atlas of the entire adult Drosophila, with male and female samples separately analyzed, using a uniform genotype and a unified single-nucleus RNA-seq (snRNA-seq) platform (11) with two

sequencing strategies: droplet-based 10x Genomics (12) and plate-based Smart-seq2 (13). The resulting Tabula Drosophilae, the first dataset within the Fly Cell Atlas (FCA) Consortium, contains more than 580,000 cells, resulting in >250 distinct cell types annotated by >100 experts from 40 laboratories. This atlas reports cellular signatures for each tissue, providing the entire Drosophila community a reference for studies that probe the effects of genetic perturbations and disease models at single-cell resolution. All data and annotations can be accessed through multiple visualization and analysis portals from https://flycellatlas.org (figs. S1 to S3). Sampling single cells across the entire adult fly

We used a unified snRNA-seq platform for all samples because it is difficult to isolate intact cells from many adult Drosophila tissues, especially cuticular ones (e.g., antenna, wing) and adipocyte-enriched ones (e.g., fat body). In addition, snRNA-seq can be applied to large multinucleated cells (e.g., muscle) and facilitates (frozen) tissue collection from different laboratories. Finally, 70 to 90% of transcriptomic information is preserved from snRNAseq compared with scRNA-seq of the same fly cell types (11). To achieve a comprehensive sampling, we used two complementary strategies. First, we dissected 12 individual tissues from both males and females as well as three sex-specific tissues (Fig. 1A). For tissues that are localized across the body (fat body, oenocytes, and trachea) and cannot be directly dissected, we used specific GAL4 lines driving nuclearÐgreen fluorescent protein (GFP) to label and collect nuclei using fluorescence-activated cell sorting (FACS). In addition, two rare cell types were sequenced only with Smart-seq2: insulinproducing cells and corpora cardiaca cells. Second, we sorted and profiled nuclei from the entire head and body, aiming to detect cell types not covered by the selected tissues. In total, we obtained 580,000 high-quality nuclei: 570,000 from 10x Genomics and 10,000 from Smart-seq2 (Fig. 1A).

1

Howard Hughes Medical Institute, Department of Biology, Stanford University, Stanford, CA 94305, USA. 2Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. 4VIB-KU Leuven Center for Brain and Disease Research, KU Leuven, 3000 Leuven, Belgium. 5 Laboratory of Computational Biology, Department of Human Genetics, KU Leuven, Leuven 3000, Belgium. 6Department of Bioengineering, Stanford University, Stanford, CA, USA. 7Department of Applied Physics, Stanford University, Stanford, CA, USA. 8Chan Zuckerberg Biohub, San Francisco CA, USA. 9Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland. 10Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland. 11Bioinformatics Competence Center, EPFL, Switzerland. 12Department of Computer Science, Stanford University, Stanford, CA 94305, USA. 13Department of Cell and Tissue Biology, University of California, San Francisco, CA 94143, USA. 14Department of Life Science, College of Natural Science, Hanyang University, 04763 Seoul, Republic of Korea. 15Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA. 16Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA. 17Aix-Marseille University, CNRS, IBDM (UMR 7288), Turing Centre for Living Systems, 13009 Marseille, France. 18Institute of Physiology and Pathophysiology, Department of Molecular Cell Physiology, Philipps-University, Marburg, Germany. 19Department of Anatomy, University of California, San Francisco, CA 94143, USA. 20Behavior and Metabolism Laboratory, Champalimaud Research, Champalimaud Centre for the Unknown, Lisbon, Portugal. 21 National Center of Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA. 22Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Kidney and Digestive Diseases, National Institutes of Health, Bethesda, MD 20892, USA. 23Centre for Neural Circuits and Behaviour, University of Oxford, Oxford OX1 3SR, UK. 24Department of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA. 25Molecular Biosciences Division, Cardiff University, Cardiff CF10 3AX, UK. 26Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 27Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA 19104, USA. 28The Penn Institute for Regenerative Medicine, Philadelphia, PA 19104, USA. 29Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA. 30Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. 31 Immunology Discovery, Genentech, Inc., South San Francisco, CA 94080, USA. 3

*Corresponding author. Email: [email protected] (N.P.); [email protected] (B.D.); [email protected] (S.R.Q.); [email protected] (L.L.); [email protected] (S.A.) †These authors contributed equally to this work. ‡Deceased. §FCA Consortium authors and affiliations are listed at the end of this paper.

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Fig. 1. Overview of the FCA. (A) Experimental platform of snRNA-seq using 10x Genomics and Smart-seq2. (B) Data analysis pipeline and data visualization using SCope (17) and ASAP (18). (C) Two versions of 10x datasets: Relaxed and Stringent. t-distributed stochastic neighbor embedding (tSNE) colors are based on gene expression: grh (epithelia, red), Mhc (muscle, green), and Syt1 (neuron, blue). The red arrow denotes an artifactual cluster with coexpression of all three markers in the Relaxed dataset. (D) tSNE visualization of cells from the Stringent 10x dataset and Smart-seq2 (SS2) cells. 10x cells

To analyze the 10x Genomics data in a reproducible manner, we used the automated VSN pipeline (14) (methods and table S1), which takes the raw sequencing data as input and performs preprocessing (e.g., normalization, doublet removal, batch-effect correction) Li et al., Science 375, eabk2432 (2022)

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are from individual tissues. Integrated data are colored by tissue (left) and platform (right). (E) Tissue-level comparison of the number of detected genes between 10x and Smart-seq2 platforms. (F) Number of cells for each tissue by 10x and Smart-seq2. Male and female cells are indicated. Mixed cells are from pilot experiments where flies were not sexed. Different batches are separated by vertical white lines. (G) All 10x cells from the Stringent dataset clustered together; cells are colored by tissue type. Tissue names and colors are indexed as in (F).

to produce LoomX-formatted files with expression data, embeddings, and clusterings (Fig. 1B and fig. S4). A presumed artifactual cluster showed expression of nearly all genes, so we added an additional preprocessing step that models and subtracts ambient RNA sig-

nals (15) to remove this cluster, resulting in a Stringent dataset of 510,000 cells (see methods and Fig. 1C). However, because adjusting the gene expression values per cell can introduce other biases (e.g., overcorrection, removal of nondoublet cells), we also retained the original 2 of 12

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Fig. 2. Cell-type annotation for dissected tissues. (A) Illustration of 15 individual tissues: Twelve are sequenced separately from males and females and three are sex-specific. Fat body, oenocyte, and tracheal nuclei were labeled using a tissue-specific GAL4 driving UASÐnuclear-GFP. (B) tSNE plot with annotations for the body wall from the Stringent 10x dataset. *1, epidermal cells of the abdominal posterior compartment; *2, epidermal cells specialized in Li et al., Science 375, eabk2432 (2022)

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antimicrobial response. (C) Uniform manifold approximation and projection (UMAP) plot with annotations for the testis from the Relaxed 10x dataset. (D) tSNE plots of the other 13 tissues from the Stringent 10x dataset. Detailed annotations are in figs. S6 to S18. (E) Number of unique annotations for each tissue. Fractions of annotated cells over all analyzed cells from the Relaxed dataset are indicated in red. 3 of 12

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Relaxed dataset of 570,000 cells. In the analyses below, unless mentioned otherwise (e.g., Fig. 2C), the Stringent dataset was used. Cells from 10x Genomics and Smart-seq2 were well integrated after batch correction using Harmony (16) (Fig. 1D). Smart-seq2 yielded a higher number of detected genes for most tissues (Fig. 1E) because cells were sequenced to a higher depth. We analyzed each tissue separately, combining the male and female runs, which yielded between 6500 (haltere) and 100,000 (head) cells and a median of 16,500 cells per tissue for 10x and between 263 (male reproductive gland) and 1349 (fat body) cells and a median of 534 cells per tissue for Smartseq2 (Fig. 1F). We obtained similar numbers of male and female cells for non–sex-specific tissues with, on average, 1895 unique molecular identifiers (UMIs) and 828 genes per cell (fig. S5). Next, all cells were combined in a metaanalysis, showing tissue-specific clusters like the germline cells of the testis and ovary and shared clusters of common cell types (Fig. 1G and figs. S24 and S25). Crowd-based cell-type annotation by tissue experts

Experts from 40 laboratories collaborated on cell-type annotation for 15 individual tissues, including 12 tissues for both sexes (antenna, body wall, fat body, haltere, heart, gut, leg, Malpighian tubule, oenocyte, proboscis with maxillary palp, trachea, and wing) and three sex-specific tissues (male reproductive gland, testis, and ovary) (Fig. 2A). We developed a consensus-voting strategy within the SCope web application (https://flycellatlas.org/scope) (17), where curators annotated clusters at multiple resolutions (ranging from 0.8 to 8; fig. S6A), with additional analysis performed in ASAP (https://flycellatlas.org/asap) (18). To ensure that cell-type annotations are consistent with previous literature and databases and to allow posteriori computational analyses at different anatomical resolutions, we used Flybase anatomy ontology terms (19). Because some cell types are annotated at low resolutions and others at high resolutions, we collapsed all annotations across resolutions and retained the annotation with the highest number of up votes. All initial annotations were performed on the Relaxed dataset and were then exported to the Stringent dataset, where field experts verified the accuracy of the annotation transfer (Fig. 2, A to E, and figs. S6 to S18). Overall, we annotated 251 cell types in the Stringent dataset (262 cell types if combining Relaxed and Stringent datasets; table S2), with a median of 15 cell types per tissue. Our dataset provides a single-cell transcriptomic profile for several adult tissues not profiled previously, including the haltere, heart, leg, Malpighian tubule, proboscis, maxillary Li et al., Science 375, eabk2432 (2022)

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palp, trachea, and wing (figs. S6 to S18). In these tissues, all major expected cell types were identified. In the proboscis and maxillary palp (fig. S7, A and B), we could annotate gustatory and olfactory receptor neurons, mechanosensory neurons, and several glial clusters. All seven olfactory receptors expressed in the maxillary palp were detected. In the wing (fig. S8), we could identify four different neuronal types—gustatory receptor neurons, pheromone-sensing neurons, nociceptive neurons, and mechanosensory neurons—as well as three glial clusters. In the leg (fig. S9), we could distinguish gustatory receptor neurons from two clusters of mechanosensory neurons. In the heart (fig. S10), we found a large proportion of resident hemocytes and muscle cells, with cardiac cells marked by the genes Hand and tinman constituting a small proportion. In the Malpighian tubule (fig. S11), 15 cell types were identified, including the different principal cells of the stellate and main segments. In the haltere (fig. S13), we identified two clusters of neurons, three clusters of glial cells, and a large population of epithelial cells. In some tissues, cell types formed a big cluster instead of being split into distinct populations. In these cases, we identified genes or pathways that showed a gradient or compartmentalized expression. For example, in the fat body (figs. S14 and S19), the main fat body cells formed one big cluster, but our metabolic pathway enrichment analysis performed through ASAP (18) revealed that fatty acid biosynthesis and degradation are in fact compartmentalized, highlighting possible fat body cell heterogeneity in metabolic capacities. Our crowd annotations with tissue experts also revealed cell types that had not been profiled previously, such as multinucleated muscle cells (Fig. 2B) and two distinct types of nuclei among the main cells in the male accessory gland (fig. S17), a cell type that was previously thought to be uniform. The high number of nuclei analyzed allowed identification of rare cell types. For example, in the testis (Fig. 2C), we identified 25 distinct cell types, covering all expected cell types, including very rare cells, such as germinal proliferation center hub cells (79 nuclei in the Relaxed version, out of 44,621 total testis nuclei). Next, we compared the distribution of cells between 10x and Smart-seq2 and found a good match based on a coclustering analysis (figs. S20 and S21). Because Smart-seq2 cells only account for a small fraction, our previous annotations focused on 10x cells. The cell-matched coclustering analysis allowed us to transfer annotations from 10x to Smart-seq2 datasets (fig. S20E), using cluster-specific markers as validation (fig. S20F). We also identified genes that were specifically detected using Smartseq2 thanks to its higher gene detection rate (Fig. 1E and fig. S20G). In summary, the high-

throughput 10x datasets form the basis for identifying cell types, whereas the Smart-seq2 datasets facilitate the detection of lowly expressed genes and enable future exploration of cell-specific isoform information. Correspondence between dissected tissues and whole head and body

To generate a complete atlas of the fly, we next performed snRNA-seq experiments on wholehead and whole-body samples. Whole-body single-cell experiments were previously performed on less complex animals (20, 21). Full head and body sequencing provides a practical means to assess the impact of mutations or to track disease mechanisms, without having to focus on specific tissues. In addition, it could yield cell types that are not covered by any of the targeted tissue dissections. In the head, we annotated 81 mostly neuronal cell types (Fig. 3A and fig. S22). In the body, we annotated the top 33 most abundant cell classes, including epithelia, muscle, and ventral nerve cord and peripheral neurons, followed by fat cells, oenocytes, germ line cells, glia, and tracheal cells (Fig. 3B and fig. S23). Many of these cell classes can be further divided into cell types for further annotation (see Fig. 2 and figs. S6 to S18). Next, we examined how well the head and body samples covered the cell types from the dissected tissues. We analyzed head, body, and tissue samples together, with most of the selected tissues clustering together with the body. We also detected head- and body-enriched clusters (Fig. 3C). One body-specific cluster contained cuticle cells, likely from connective tissue (Fig. 3D). Others were relatively rare cell types in their respective tissues, such as adult stem cells. Conversely, most tissue clusters contained body cells, with only a small number being completely specific to dissected tissues. Because tissue-specific clusters were mostly observed in tissues with high cell coverage, such as the testis and Malpighian tubule, we anticipated that these clusters would also be identified in the body upon sampling a larger number of cells. For the head, the antenna and proboscis with maxillary palp were dissected for tissue sequencing. Cell types from those two tissues largely overlapped with head cells. Many other cell types, such as central brain cells, including Kenyon cells (ey, prt) and lamina glia (repo, Optix), were only detected in the head sample. To compare our data with existing datasets, we integrated our head snRNA-seq dataset (“head” hereafter) with published brain scRNAseq data (“brain” hereafter) (17, 22–24) (Fig. 3E). Head-specific clusters made up 20% of the cells, including the antennae, photoreceptors, muscle, cone cells, and cuticular cell types, whereas the other 80% were present in clusters containing both head- and brain-derived 4 of 12

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Fig. 3. Whole-head and whole-body sequencing leads to full coverage of the entire fly. (A) tSNE of the whole-head sample with 81 annotated clusters. See fig. S22 for full cell types. Many cells in the middle (gray) are unannotated, most of which are central brain neurons. (B) tSNE of the whole-body sample with 33 annotated clusters, many of which can be further divided into subclusters. Cells in gray are unannotated. See fig. S23 for full cell types. (C) tSNE of the entire dataset colored by standardized tissue enrichment, leading to the identification of head- and body-specific clusters, is shown on the left. Stacked bar plot showing tissue composition (head, body, or dissected tissues) for different Li et al., Science 375, eabk2432 (2022)

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clusters at Leiden resolution 50 are shown on the right. (D) Examples of headand body-specific clusters. (E) Integration of a brain scRNA-seq dataset with the head snRNA-seq for label transfer. Outlined are example clusters revealed by the head snRNA-seq dataset but not by the brain scRNA-seq datasets, including epithelial cells (EPI), photoreceptors (PRs), olfactory receptor neurons (ORNs), and muscle cells (MUS). (F) Subclustering analysis reveals types of photoreceptors, including inner and outer photoreceptors, with the inner photoreceptors further splitting into R7 and R8 types, and mushroom body Kenyon cells comprising three distinct types: a/b, a′/b′, and g. 5 of 12

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cells covering the neuronal and glial cell types of the brain. This coclustering across genotypes and protocols underscores the quality and utility of our snRNA-seq data compared with that of scRNA-seq data. Next, we used machine-learning models to predict annotations per cluster, followed by manual curation (22). Given the high number of neuron types, additional subclustering was performed on each cluster, identifying subtypes of peptidergic neurons (dimm, Pdf) and olfactory projection neurons based on oaz, c15, and kn. Finally, we identified many cell types in the optic lobe, including lamina (e.g., L1 to L5), medulla (e.g., Mi1, Mi15), lobula (e.g., LC), and lobula plate (e.g., LPLC). Using acj6 and SoxN, we identified the T4/T5 neurons of the optic lobe that split in T4/T5a-b and T4/T5c-d subtypes by subclustering. A big clump of neurons remained unannotated (Fig. 3A), indicating that our dataset cannot resolve the complexity of the central brain, which may contain hundreds to thousands of neuron types. Subclustering in the combined dataset separated inner and outer photoreceptors from the dorsal rim area and ocellar photoreceptors, with the inner photoreceptors further splitting into R7 and R8 types, each with pale and yellow types based on rhodopsin expression (Fig. 3F). Additionally, Kenyon cells were split into three types: a/b, a′/b′, and g (17). These cases highlight the resolution in our dataset and the potential of using subclustering to discover rare cell types. Cross-tissue analyses allow comparison of cell types by location

Using the whole-body and whole-head sequencing data, we assigned cells to major cell classes (e.g., epithelial cells, neurons, muscle cells, hemocytes), which allowed us to compare common classes across tissues (Fig. 4, A to C, and figs. S24 and S25). First, we compared blood cells across tissues by selecting all Hml-positive cells, a known marker for hemocytes (Fig. 4D). Combining hemocytes across tissues revealed a major group of plasmatocytes, the most common hemocyte type (~56%), crystal cells (1.5%; PPO1, PPO2), and several unknown types (fig. S26, A and B). Looking deeper into the plasmatocytes, we uncovered gradients based on the expression of Pxn, LysX, Tep4, trol, and Nplp2 that can be linked to maturation and plasticity, with Pxn-positive cells showing the highest Hml expression, whereas Tep4, trol, and Nplp2 are prohemocyte markers (25). Furthermore, different antimicrobial peptide families such as the Attacins and Cecropins were expressed in different subgroups, indicating specialization. Finally, expression of acetylcholine receptors was specific for a subset of hemocytes, relating to the cholinergic anti-inflammatory pathway as described in humans and mice (26). Lamellocytes were not Li et al., Science 375, eabk2432 (2022)

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observed in adults as previously suggested (27). On the contrary, an unknown hemocyte type expressed Antp and kn (43 cells, 0.5%) reminiscent of the posterior signaling center in the lymph gland, an organization center previously thought to be absent in the adult (28, 29) (fig. S26B). These findings highlight the value of performing a whole organism– level single-cell analysis and constitute a foundation for investigating the fly immune system in greater detail. Second, we compared the muscle cells of the different tissues (Fig. 4E and fig. S26, C and D). Muscle cells are syncytia—individual cells containing many nuclei—and to our knowledge have not been profiled by single-cell sequencing before our study. With snRNA-seq, we recovered all known muscle cell types, with specific enrichment in the body, body wall, and leg. This comprehensive view of the fly muscular system highlights a separation of visceral, skeletal, and indirect flight muscle based on the expression of different troponins. Specifically, we discovered gradients of dysf and fln in the indirect flight muscle, which may indicate regional differences in these very large cells (>1000 nuclei) (fig. S26E). We identified four types of visceral muscle in the gut based on expression of the AstC, Ms, Dh31, and CCAP neuropeptide receptors, indicating potential modulators for muscle contraction (30). Ms and Dh31 have been described to function in spatially restricted domains (30–32), suggesting similar domains for AstC and CCAP. All visceral muscle cells are enriched for the receptor of Pdf, a neuropeptide involved in circadian rhythms, pointing toward a function in muscle contraction as well (33). Transcription factors and cell-type specificity

Our data allow the comparison of gene expression across the entire fly. Clustering cell types showed the germline cells as the most distinct group, followed by neurons (figs. S27 to S32). We calculated marker genes for every cell type using the whole FCA data as background, with 14,240 genes found as a marker for at least one cell type and a median of 638 markers per cell type [minimum: visceral muscle (94); maximum: spermatocyte (7736)]. Notably, markers specific for cell types in a tissue were not always specific in the whole body (fig. S33). Next, we calculated the tau score of tissue specificity (34) for all predicted transcription factors (TFs) (3) and identified 500 TFs with a score >0.85, indicating a high specificity for one or very few cell types (Fig. 5A and table S3). Of these TFs, 127 were “CGs” (computed genes), indicating that their functions are poorly studied. We found that the male germline stands out in showing expression of a great number of cell type–specific TFs. This may be related to the broad activation of many genes in late spermatocytes, as discussed below.

Similar analysis across broad cell types (Fig. 5, B and C) identified 156 TFs with high tau scores, for example, the known regulators grh for epithelial cells and repo for glia, as well as 24 uncharacterized genes. Network visualization shows the grouping of central nervous system (CNS) neurons and sensory organ cells, including many sensory neurons, with shared panneuronal factors such as onecut and scrt but with each cluster having a distinct set of TFs, such as ey, scro, and dati for CNS neurons and lz and glass (gl) for sensory neurons. In addition to the specificity of TF expression, we predicted gene regulatory networks based on coexpression and motif enrichment using SCENIC (31). Because of the stochasticity of this network inference method, we ran SCENIC 100 times, ranking predicted target genes by their recurrence. This approach selected 6112 “regulons” for 583 specific TFs across all tissues, whereby each regulon consists of the TF, its enriched motif, and the set of target genes that are predicted in at least 5 out of 100 runs. In fat cells, our analysis predicted a regulon for sugarbabe (sug), a sugar-sensitive TF necessary for the induction of lipogenesis (32). In photoreceptors, the analysis identified a gl regulon, with key photoreceptor markers such as Arr1, eya, and multiple rhodopsins as predicted target genes (Fig. 5, D and E) (33). The SCENIC predictions for all cell types are available through SCope (https://flycellatlas.org/scope). A comparative analysis of genes across broad cell types or tissues (Fig. 5F and fig. S34) identified common genes and specifically expressed genes, such as a shared set of 555 housekeeping genes that are expressed in all tissues. The testis has the highest number of specifically expressed genes consistent with previous reports (34), followed by the Malpighian tubule and male reproductive glands (fig. S34). These tissue-specific genes seemed to be evolutionarily “younger” based on GenTree age compared with the set of commonly expressed genes that are all present in the common ancestor. This suggests that natural selection works on the tissue specialization level, with the strongest selection on testis, male reproductive tract, and Malpighian tubules (35). In addition, this analysis allowed an estimation of transcriptomic similarity or difference measured by the number of shared distinct genes. For example, the two flight appendages, the haltere and wing, share a set of 16 specifically expressed genes, reflecting the evolutionary origin of halteres as a modified wing (36) (fig. S34). Analysis of sex-biased expression and sex-specialized tissues

To study sex-related differences, we compared male- and female-derived nuclei for all common tissues (fig. S35) and found roX1/2 and Yp1/2/3 as the top male- and female-specific genes, respectively. Notably, a large fraction 6 of 12

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Fig. 4. Cross-tissue analyses of common cell classes. (A) Overview of main cell classes identified throughout the fly cell atlas. Male repr. syst. and fem. repr. syst., male and female reproductive system; male germ. cell and fem. germ. cell, male and female germline cells. (B) tSNE plots showing expression of four markers in four common cell classes. (C) Composition of whole-head and whole-body samples, showing a shift from neurons to epithelial and muscle cells. Composition of the entire FCA shows enrichment for rarer cell classes compared with the whole-body sample. (D) Cross-tissue analysis of hemocytes reveals different cell states of plasmatocytes. Annotations marked in blue are hemocytes containing markers of different cell types, including lymph gland posterior signaling center (LGP), muscle (MUS), antenna (ANT), neurons (NEU), photoreceptor (PR), male accessory glands (MAG), glia (G), male testis and spermatocyte (MS), odorant-binding proteins (OBP), and heat-shock proteins (Hsp). Other abbreviations show top marker gene(s) in Li et al., Science 375, eabk2432 (2022)

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red. Plasmatocytes and crystal cells are indicated. On the right are genes showing compartmentalized expression patterns within the plasmatocyte cluster. (E) Cross-tissue analysis of muscle cells reveals subdivision of the visceral muscle cells based on neuropeptide receptors. Annotations marked in blue are muscle cells containing markers of different cell types, including neuron (NEU) and male testis and spermatocyte (MS). Muscle cells from three body parts are indicated: head muscle (HEAD), body muscle (BODY), and testis muscle (TESTIS). Other annotated muscle types include indirect flight muscle (IFM), ovarian sheath muscle (OSM), abdominal visceral muscle (ABD), dpy expressing muscle (DPY), visceral muscle of the midgut AstC-R2 (VMM-A), visceral muscle of the crop MsR1 (VMC-M), visceral muscle of the midgut Dh31-R (VMM-D), and visceral muscle CCAP-R (VM-C). Pdfr is expressed in all visceral muscle cells, including the ovarian sheath muscle; the other four receptor genes (AstC-R2, MsR1, Dh31-R, CCAP-R) are expressed in different gut visceral muscle types. 7 of 12

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Fig. 5. TF pleiotropy versus cell-type specificity. (A) Heatmap showing the expression of key marker genes and distinctive TF profiles for each of the annotated cell types. TFs were selected based on tau score. Cell types were grouped based on hierarchical terms: CNS neurons (N), sensory organ cells (S), epithelial cells (E), muscle cells (M), glia (G), fat cells (F), oenocytes (O), hemocytes (H), (fe)male reproductive system and germline (MR, MG, FR, FG), excretory system (X), tracheal cell (T), gland (L), cardiac cell (C), and somatic precursor cell (P). (B) A network analysis of TFs and cell classes based on similarity of ontology terms, reveals specific and shared TFs across the individual tissues. (C) Heatmap showing the expression of specific TFs per cell class. Factors from the literature are highlighted. (D) Glass is specifically expressed in photoreceptors and cone cells in the head. (E) Overview of the Glass regulon of 444 target genes, highlighting known photoreceptor marker genes. (F) Gene expression comparison across broad cell types. Only sets with more than 10 genes are shown. The left bar graph shows the number of uniquely expressed genes for each tissue. The top bar graph shows the gene age in branches, ranging from the common ancestor to D. melanogasterÐ specific genes (http://gentree. ioz.ac.cn). See fig. S34 for a tissue-based comparison.

of genes with male-enriched expression were uncharacterized (37). The primary sex-determination pathway in somatic cells leads to sex-specific splicing of doublesex (dsx) to encode femaleor male-specific TFs (38) (Fig. 6A). Consistent with this, we found dsx expression in a largely non–sex-specific pattern, whereas many other genes showed sex-biased expression (Fig. 6B). Next, we performed differential expression between sexes for all cell types. Notably, cell types tended to show either high female or male bias, but not both (Fig. 6, B and C). We Li et al., Science 375, eabk2432 (2022)

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found strong female bias in the excretory system, including the principal and stellate cells of the Malpighian tubule and in the pericardial nephrocytes (Fig. 6C). Female-biased genes (i.e., Ics and whe) were differentially expressed under high-salt conditions, suggesting sex-bias in nephric ion transport. Across cell types, sexbiased expression strongly correlated with dsx expression (Fig. 6D) (39), consistent with the role of Dsx as a key regulator. Among all tissues in the adult fly, those best characterized that have ongoing cellular dif-

ferentiation are the gut, ovaries, and testis. Trajectory analysis has been performed on the gut and ovary stem cell lineages in previous studies (40–42), and our FCA data on gut and ovary accurately coclustered with these published datasets (figs. S36 and S37). Therefore, we focused on the testis plus seminal vesicle as a case study. The testis has two populations of stem cells, the somatic cyst stem cells (CySCs) that produce cell types with supporting roles essential to spermatogenesis, and the germline stem cells (GSCs) that produce haploid 8 of 12

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Fig. 6. Sex-biased expression and trajectory analysis of testis cell lineages. (A) Simplified sex-determination pathway. Sex-chromosome karyotype (XX) activates Sex-lethal (Sxl), which regulates transformer (Tra), resulting in a female Dsx isoform (DsxF). In XY (or X0) flies, Sxl and Tra are inactive (light gray) and the malespecific DsxM is produced. (B) dsx expression and female- and male-biased expression projected onto tSNE plots of all female (top left) and male (top right) cells except reproductive tissue cells (tables S4 and S5). Female-biased (bottom left) and male-biased (bottom right) expression measured as the percentage of genes in the cluster showing biased expression in favor of the respective sex (table S6) are also shown. The percentage values were computed for each annotated cluster, and those cluster-level values were projected onto the individual cells in the corresponding clusters. For all four tSNE plots, values outside the scale in the heatmap key are represented by the closest extreme color (> and < signs in the scale). (C) Scatter plot of femaleand male-bias values across nonÐreproductive cell clusters defined as percentage of sex-biased genes (at least twofold change with false discovery rate 2 cm from any radiographic abnormality on magnetic resonance imaging, showed no abnormalities on routine electrocorticography, and were histologically normal on a rapid hematoxylin and eosin stain. Diagnosis of human brain AVMs 6 of 12

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Condition

UMAP

AVM Control

Immune

UMAP

UMAP

pvM * pvM 1 pvM 2 ExV Mo1 Mo2 Mo3 MG cDC pDC CD4 TC Treg CD8 TC1 CD8 TC2 NK BC Div

UMAP

C

Treg (0.7%)

pvM * (15.9%)

CD4 TC (16.3%)

CD4 TC (19.2%)

cDC pvM 2 (4.5%) (4.3%)

2.5

0

% Exp. 0 25 50 75

Condition

D pvM * (11.4%)

AVM

AVM

cDC (5.3%)

Control

Myeloid

AVM (% Compositon) Total cells = 41,207

ns

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20

30

40

AVM

i.

15000

IBA1+ P2RY12

**

10000

**

0.2

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-3 0 3 log2(cell ratio per image)

IBA1+ P2RY12

32

6

**

5 0

4

Distance ( m)

IBA1+ P2RY12+ 10

128 log2(counts)

Distance ( m)

256

2

log (counts)

ii.

32

80

ns

40 0

8

2 0

Fig. 4. Cerebrovascular inflammation with malformation. (A) UMAP visualizations of coembedded immune cells states in control cerebrovasculature and brain AVMs (n = 5 donors per condition). (Top) Colored by condition. Control, gray; AVM, red. (Bottom) Colored by cell state. pvMf perivascular macrophage; pvMf*, activated perivascular macrophage; Mo, monocyte; MG, microglia; ExV, ex vivo activated myeloid cells; cDC, conventional dendritic cells; pDC, plasmacytoid dendritic cells; CD8 TC, CD8+ T cells; CD4 TC, CD4+ T cells; Treg, regulatory T cells; NK, natural killer cells; BC, B cells; and Div, dividing immune cells. (B) Dot plot showing expression of immune cell markers. (C) Pie charts showing immune cell state proportions in (left) controls and (right) AVMs. (D) Bar graph with individual data points showing the proportion of myeloid and lymphoid immune cells captured in AVMs (red) and controls (gray). n = 5 donors per condition, mean ± SEM,

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IBA1+ P2RY12+

5000

Podocalyxin SMA IBA1 P2RY12 DAPI

Winkler et al., Science 375, eabi7377 (2022)

1.0

0.4

-6

i.

0.5

Immune cell proportion (%)

Control

IBA1+ P2RY12+

Density

Cell number per mm3

IBA1+ P2RY12

i.

0

Condition

Arteriovenous Malformation Nidus

ii.

50

% of cells

G

F

Control

pvM * BC cDC CD8 TC1 CD8 TC2 Div ExV MG Mo1 Mo2 Mo3 NK pDC pvM 1 pvM 2 CD4 TC Treg

BC (1.3%)

CD8 TC1 (14.4%) pvM 1 CD8 TC1 (12.6%) CD8 TC2 Lymphoid (12.8%) (2.3%) pDC CD8 TC2 (0.9%) Div ExV NK (3.9%) (6.0%) (0.7%) (2.1%) Mo3 Mo1 Div (4.7%) Mo2 0 MG (6.2%) (5.9%) (0.4%) (1.6%)

pDC (0.4%) NK (5.0%) Mo3 (2.6%) Mo1 ExV Mo2 (4.3%) MG (7.0%) (2.1%) (3.3%)

E

Condition

*

Control (% Compositon) Total cells = 14,408

Avg. Exp.

ExV MG pvM * pvM 2 pvM 1 Mo3 Mo2 Mo1 cDC pDC Div NK CD8 TC2 CD8 TC1 Treg CD4 TC BC

BC (5.4%)

pvM 2 (2.2%) pvM 1 (13.1%)

Treg (1.0%)

B

CD79B IGLC2 CD3D CCR7 IKZF2 IL2RA CD8A CD8B GZMB GNLY MKI67 TOP2A JCHAIN CD1C EREG KYNU LYZ S100A8 GPNMB CTSB LYVE1 MRC1 SPP1 TREM2 CCL3 IL1B P2RY12 CX3CR1 JUNB HSPA1A AIF1

A

50

100 Distance ( m)

150

0

50

100 150 Distance ( m)

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250

two-tailed t test. *P < 0.05; ns, not significant. (E) Bar graph of relative immune cell state proportions normalized to total cells sequenced in AVMs (red) and controls (gray). (F) Representative confocal microscopy analysis of endothelial cells [cyan, podocalyxin (PODOXL)], smooth muscle cells [blue, a smooth muscle actin (SMA)], macrophages [magenta, ionized calcium binding adaptor molecule 1 (IBA1) encoded by the gene AIF1 in (B)], and microglia [green, purinergic receptor P2Y12 (P2Y12)]. Scale bar, 1 mm. (i) Layered ameboid perivascular macrophages. (ii) Perivascular microglial response. Scale bars, insets, 50 mm. (G) Quantification of (top left) abundance, (top right) cell ratio per image, and (bottom) perivascular distance of IBA1+P2R12− macrophages and IBA1+P2R12− microglia (n = 3 donors per condition; three nonadjacent sections per donor; 8 to 10 images per section). Mean ± SEM, two-tailed t test. **P < 0.01; ns, not significant.

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Ruptured AVMs M o3

B

TC SM C 7

PV

5 C

N d2

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2 M PV

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SM C 6

SM C 8

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up tu r up ed tu re d

GPNMB - Mo

-

GPNMB + Mo

R

nr Laminin

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Cocktail

RGD

CD44

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Shape Shared Incoming Outgoing Bidirectional

SPP1

1

ns ns

THBSAnnexin ANGPTL CCL 0 MIF

125 **

CC3+ cells per 103 SMCs

Differential Incoming Interaction Strength

Collagen

Vehicle

50

CC3 DAPI

Shared AVM CTRL

SM

CC3+ cells

per 103 SMCs

4000

U

G Color

2

M

PC

*

0

F

GPNMB- Mo GPNMB+ Mo

No Mo CC3 DAPI

3 + GPNMB cells per mm

M

o1 M

E 8000

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N d N 1 K o2

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*

D

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*

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-3

C

1

A

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scRNAseq reference

1

BC C TL

Unruptured AVM (n =13)

Cell proportions

*

2

Ar t Ar 1 t2

Rupture gene module

3. Cell-specific Gene expression

Change in Cell Proportion (T-Statistic)

2. In silico cell deconvolution

1. Bulk RNAseq differential expression

rt

3

Ruptured AVM (n =26)

**

3

A

MHC−II FN1NOTCH -1

VISFATIN APP CD45 0

2

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6

Fig. 5. Cell states implicated in brain AVM rupture. (A) Cellular deconvolution and cell-specific differential expression analysis of bulk RNA-seq from ruptured and unruptured brain AVM. (B) Bar graph of change in cell proportion t statistic in ruptured AVMs. Purple, increased cell abundance; blue, decreased cell abundance. *P < 0.05; **P < 0.01. (C) Representative confocal microscopy imaging showing GPNMB+ monocytes (green), endothelial cells [cyan, podocalyxin (PODOXL)], and smooth muscle cells [magenta, aÐsmooth muscle actin (SMA)] in unruptured and ruptured AVMs. Scale bar, 100 mm. (D) Quantification of GPNMB+ monocytes in unruptured (blue) and ruptured (purple) AVMs (n = 3 donors per condition; three nonadjacent sections per donor; 8 to 10 random images per section). Mean ± SEM, two-tailed t test. *P < 0.05. (E) (Top) Confocal microscopy analysis of cleaved caspase-3+ (green, CC3) human primary SMCs after coculture with GPNMB+ and GPNMB− monocytes isolated from ruptured AVMs. DAPI (magenta) stains are cell nuclei. White, colocalization of CC3 and DAPI; arrow, CC3+ cell.

were angiographically confirmed preoperatively, and fresh tissues were acquired as part of planned surgical resection. For scRNA-seq, only unruptured brain AVMs were enrolled into the study. Specimen orientation was mainWinkler et al., Science 375, eabi7377 (2022)

**

75 50 25 0

-

+

-

+ +

-

+

-

+

+

-

+

Scale bar, 20 mm. (Bottom) Quantification of CC3+ smooth muscle cells (n = 3 independent cultures per condition). Mo, monocytes. Mean ± SEM, ANOVA with Tukey post hoc test. *P < 0.05; ns, not statistically significant. (F) Scatterplot of dysregulated cell communication pathways in AVM GPMNB+ monocytes (Mo3) relative to controls by scRNA-seq. Red, up-regulated in AVM; blue, up-regulated in control; gray, shared between conditions; triangle, outgoing network; square, incoming network; and diamond, outgoing and incoming network. (G) (Top) Confocal microscopy analysis of cleaved caspase-3+ (green, CC3) human primary smooth muscle cells treated with osteopontin (OPN, encoded by SPP1) and CD44-neutralizing antibody, RGD integrin inhibitor, or inhibitor cocktail. DAPI (magenta) stains are cell nuclei. White, colocalization of CC3 and DAPI; arrow, CC3+ cell. Scale bar, 20 mm. (Bottom) Quantification of CC3+ smooth muscle cells (n = 5 to 6 independent cultures per condition). Mo, monocytes. Mean ± SEM, ANOVA with Tukey post hoc test. **P < 0.01; ns, not statistically significant.

tained to ensure coverage of arteriovenous axis with surgical clips of different sizes with aide of intraoperative fluorescent angiography. All tissues were acquired in close collaboration with neurosurgeons trained in tissue isolation

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**

100

OPN CD44 RGD CD44 + RGD

Differential Outgoing Interaction Strength

**

techniques to minimize tissue disruption such as avoidance of electrocautery. For bulk sequencing experiments, we utilized snap-frozen ruptured and unruptured AVM specimens as part of our biorepository. Patient demographic 8 of 12

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information for all tissues utilized is summarized in table S1. Isolation of cerebrovascular specimens

No differences in vascular isolation methods were applied between normal cerebral cortex or AVM tissues. Tissue specimens were placed in chilled preoxygenated Dulbecco’s modified Eagle medium (DMEM, Fisher Scientific, Waltham, MA, catalog number: MT10017CV) and transported to the laboratory on ice. All tissue handling was performed with autoclavesterilized equipment within a class II biological safety cabinet. Large arteries and veins were selectively isolated under 5× magnification with a Leica MZ75 dissecting microscope (Leica Microsystems, Wetzlar, Germany) with two #5/45 Dumont micro-forceps (Fine Science Tools, Foster City, CA, catalog number: 11251-35). Under 5X magnification, the lumen of each vessel was longitudinally opened with a #15 scalpel blade (Fine Science Tools) or flushed with DMEM. The large vessels were placed in an Eppendorf LoBind 5-ml tube (Eppendorf North America, Enfield, CT, catalog number: 0030122348) and kept on ice in chilled oxygenated DMEM. Following removal of all visible vasculature, the microvasculature was isolated using dextran gradient centrifugation followed by cell-strainer filtration (20–22). More specifically, tissue was cut into ~1- to 2-mm pieces with a scalpel and gently homogenized in a Dounce homogenizer containing oxygenated pre-chilled DMEM with 1% bovine serum albumin (BSA) (Sigma Aldrich, St. Louis, MO, catalog number: A9418). The homogenate was mixed in 18% dextran solution (MW: ~70,000 Da; Sigma-Aldrich, catalog number: 31390) at a volume ratio of 1:1 and centrifuged at 6000g for 20 min at 4°C. This resulted in a microvascular pellet and floating vasculardepleted brain. The floating vessel-depleted brain was gently aspirated and discarded. The vascular pellet resuspended in DMEM with 1% BSA and passed through a 40-mm cell strainer (Fisher Scientific, catalog number: 08-771-1) to remove circulating cells or other debris. Microvascular fragments remain trapped on top of the cell strainer and were subsequently collected by inverting with cell strainer and washing with prechilled oxygenated DMEM. A small aliquot was visualized at 10X magnification to confirm both purity and yield with brightfield microscopy. The microvascular fragments were pelleted by centrifugation at 500g for 5 min. The supernatant was aspirated and then pooled with the microdissected arteries and veins from the same individual in prechilled oxygenated DMEM. These pooled preparations are referred to as isolated vascular preparations for subsequent steps and maintained in chilled oxygenated media on ice and immediately processed to create single-cell suspensions. Winkler et al., Science 375, eabi7377 (2022)

Generation of vascular single-cell suspensions

Isolated vascular preparations were incubated for 45 min in 0.2% collagenase type 2 (Worthington Biochemical Corporation, Lakewood, NJ, catalog number: LS004176) diluted in preoxygenated DMEM at 37°C with gentle agitation in an Eppendorf LoBind 5-ml tube (Eppendorf North America). Cell suspensions were filtered through a sterile 40-mm cell strainer (Fisher Scientific) to isolate undigested debris. Cells contained within the flowthrough were collected by centrifugation at 500g at 5 min and the supernatant carefully aspirated. To lyse any residual erythrocytes, the cell pellet was resuspended in Gibco ACK lysing buffer (Fisher Scientific, catalog number: A1049201) for 3 min at room temperature. Cells were then collected by centrifugation at 500g at 5 min and washed three times with sterile RNase-free phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537500ml) containing 0.04% BSA. Cells were pelleted with centrifugation at 500g for 5 min and resuspended in PBS with 0.04% BSA. To confirm cell viability and yield, a 10-ml aliquot of the cell suspension was mixed 1:1 with 0.4% trypan blue (Thermo Fisher Scientific, catalog number: T10282) to stain non-viable cells. Cells were then counted on a hemocytometer. scRNA-seq

All scRNA-seq experiments were performed on freshly isolated, whole cells as described above. Droplet-based scRNA-seq was performed with 10X Genomics Chromium Single Cell 3 prime reagent kits v3 as described by the manufacturer (10X Genomics, Pleasanton, CA, product code: 1000092; n = 5 normal cortex samples and n = 5 AVMs). Based on hemocytometer counts, single cells were loaded onto chromium chips with a capture target of 15,000 cells per sample. When cell yield was sufficient, two reactions per individual were performed. Libraries were prepared following the provided manufacturer’s protocol. Quality of prepared sequencing libraries were confirmed by electrophoretic analysis on an Agilent 4200 TapeStation System (Agilent Technologies, Santa Clara, CA). Libraries were sequenced with an Illumina NovaSeq 6000 (Illumina, San Diego, CA) with a targeted sequencing depth of 50,000 reads per cell. Bulk RNA sequencing

Undigested, isolated vascular tissues from ruptured and unruptured AVMs were obtained from the operating room, snap frozen in liquid nitrogen and stored at −80°C. Snap frozen tissues were embedded in Optical Cutting Temperature Compound (Sakura Finetek USA, Torrance, CA, catalog number: 4583) and cryosectioned at a thickness of 20 mm. Tissue scrolls were collected in RNAase free Eppendorf LoBind-1.5 ml microcentrifuge tubes

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(Eppendorf North America, catalog number: 022431021). DNA/RNA Shield reagent (Zymo Research, Irvine, CA, catalog number: R1100) was added. Tissues were mechanically homogenized with a Squisher-Single homogenizer (Zymo Research, catalog number: H1001) and subsequently digested with proteinase K (Zymo Research, catalog number: R1057). Total RNA was isolated from the homogenized tissues with the Quick-RNA Miniprep Plus Kit as instructed by the manufacturer (Zymo Research, catalog number: R1057). The purified RNA was quantified with a NanoDrop (Thermo Fisher Scientific) and integrity confirmed with an Agilent 4200 TapeStation System (Agilent Technologies, Inc.). Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit as instructed by the manufacturer (New England Biolabs, Ipswich, MA, catalog number: E6310X), and sequencing libraries prepared with the SEQuoia Complete Stranded RNA Library Prep Kit (BioRad Laboratories, Hercules, CA, catalog number: 17005710). Sequencing library quality was confirmed on an Agilent 4200 TapeStation System (Agilent Technologies, Inc) and quantified with a Qubit dsDNA high sensitivity assay kit (Thermo Fisher Scientific). Libraries were sequenced in batch with an Illumina NovaSeq 6000 sequencer (Illumina) with targeted read depth of at least 5 × 107 reads per sample. Rebus Esper spatial omics platform

Spatially resolved, multiplexed in situ RNA detection and analysis was performed using the automated Rebus Esper spatial omics platform (Rebus Biosystems, Santa Clara, CA). By intersecting a list of known or candidate cell type markers generated by scRNA-seq, suitability for probe design, including gene length and relative abundance, and design constraints for compatibility with the Rebus Esper spatial omics platform using proprietary software, we generated the following gene probe panel: MECOM, RGS16, RARA, MFSD2A, TAGLN, IL1R1, VEGFC, KCNJ8, DCN, TMEM132C, CCL19, CLDN5, PDGFRB, HIGD1B, KCNT2, ALDH1A2, and ALDH1A1. Experiments and analyses using the Rebus Esper spatial omics platform were performed as previously described (70). Immunofluorescent staining of tissues and cerebrovascular fragments

Formalin-fixed paraffin-embedded tissue sections were cut at a thickness of 6 mm, deparaffinized with xylene, and rehydrated to distilled water with serial ethanol washes. For immunostaining of isolated vessel fragments, cerebrovascular vessel isolation was performed as described above and immersion fixed in 4% paraformaldehyde overnight at 4°C. For antigen retrieval, all specimens were incubated with pH 9 Tris-EDTA buffer at 97°C for 15 min. Tissue sections were then blocked with PBS 9 of 12

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containing 0.2% gelatin, 1% donkey serum, and 1% triton for 1 hour at room temperature and incubated in the following primary antibodies overnight at 4°C. Primary antibodies included: podocalyxin (1:100, R&D Systems, Minneapolis, MN, catalog number: AF1658), alpha smooth muscle actin clone 1A4 (1:100, Dako North America, Inc. Carpinteria, CA, catalog number: M085129), P2RY12 (1:500, Sigma Aldrich, catalog number: HPA014518), IBA1 (1:500, Synaptic Systems, Goettingen, Germany, catalog number: 234004), PECAM1 (1:50, Agilent Technologies, catalog number: M0882329), PLVAP (1:100, Sigma Aldrich, catalog number: HPA002279), angiopoietin-2 (1:100, R&D systems, catalog number: AF623), PDGFRB (28E1) (1:100, Cell Signaling Technology, catalog number: 3169S), GPNMB (1:100, R&D systems, catalog number: AF2550), and CD8 clone C8/ 144B (1:100, Dako North America, catalog number: M710301). Sections were washed with PBS containing 1% Triton and incubated with fluorescent dye–conjugated secondary antibodies for 2 hours at room temperature. Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody (1:500, Thermo Scientific, catalog number: A32766), Alexa Fluor 546conjugated donkey anti-rabbit secondary antibody (1:500, Thermo Fisher, catalog number: A10040), and Alexa Fluor 647-conjugated donkey anti-goat secondary antibodies (1:500, Thermo Fisher, catalog number: A32787) were used to detect mouse, rabbit, and goat primary antibodies, respectively. Sections were washed and autofluorescence quenched by incubating with 1% Sudan Black (Sigma Aldrich, catalog number: 199664) for 10 min at room temperature. Slides were mounted with 4-prime,6diamidino-2-phenylindole (DAPI)–containing Fluoromount-G (SouthernBiotech, Birmingham, AL, catalog number: 0100-20). All imaging was performed with a Lecia TCS SP8 X confocal microscope with a 20X objective (Leica Microsystems). Fluorescence-activated cell sorting (FACS)

To isolate circulating GPNMB+ and GPNMB− monocytes, 10 ml of whole blood was collected in standard 5-ml EDTA-containing vacutainer blood collection tubes obtained from adult patients with acutely ruptured AVMs. Cells were subsequently centrifuged at 500g for 5 min and erythrocytes were then lysed with incubation in Gibco ACK lysing buffer (Fisher Scientific, catalog number: A1049201). The resulting cell suspension was filtered through a sterile 40-mm filter to remove debris and washed with PBS containing 0.04% BSA. Cell suspensions were then resuspended in FACS staining buffer (Thermo Fisher Scientific, catalog number: 00-4222-26). Cells were blocked to prevent non-specific staining, and cells were stained with Alexa Fluor 647-conjugated mouse anti-human CD45 monoclonal antiWinkler et al., Science 375, eabi7377 (2022)

body clone HI30 (1.25 ng/ml, Thermo Fisher Scientific, catalog number: 51-0459-42), FITCconjugated mouse anti-human CD11b monoclonal antibody clone ICRF44 (5.0 ng/ml, Thermo Fisher Scientific, catalog number: 11-0118-42), and PE-conjugated mouse antihuman GPNMB monoclonal antibody clone HOST5DS (1.25 ng/ml, Thermo Fisher Scientific, catalog number: 12-9838-42) for 30 min at room temperature. Cells were subsequently isolated by FACS with a BD FACSAria II Flow Cytometer (BD Biosciences, Franklin Lakes, NJ). Viable CD45+ CD11b +GPNMB + cells and CD45+CD11b+GPNMB− cells were separately collected for subsequent coculture experiments. Cell culture

All cell culture experiments utilized primary human brain vascular smooth muscle cells (VSMCs, ScienCell Research Laboratories, Carlsbad, CA, catalog number: 1100). Cells were cultured in SMC media in 5% CO 2 at 37°C. Early passage (P3, P4) cultures were used in the present study. Primary VSMCs were plated in equal number for all conditions. VSMCs were grown on 96-well tissue culture plates pre-coated with collagen. For coculture experiments, CD45+CD11b+GPNMB+ or CD45+CD11b+GPNMB− cells were immediately cocultured with VSMCs following FACS isolation at an approximate ratio of 1:1 (71). Monocytes and VSMCs were cocultured for 24 hours and then subsequently fixed with 4% paraformaldehyde for subsequent immunostaining. For osteopontin (OPN, encoded by SPP1) treatment studies, cells were pretreated with vehicle control or anti-human CD44 neutralizing monoclonal antibody (10 mg/ml, Thermo Fisher Scientific, catalog number: MA4400), RGD peptide to inhibit integrin receptors (10 mM, Sigma Aldrich, catalog number: A8052), or both in combination for 30 min as previously described (65). Cultures were then subsequently treated with OPN (200 ng/ml, Sigma Aldrich, catalog number: SRP3131) for 6 hours. The cell culture media was changed and cells were then fixed with 4% paraformaldehyde for subsequent immunostaining. Cell culture immunostaining

Paraformaldehyde fixed cells were blocked with PBS containing 0.2% gelatin, 1% donkey serum, and 0.1% triton for 1 hour at room temperature and incubated in primary antibodies overnight at 4°C. Primary antibodies included: alpha smooth muscle actin clone 1A4 (1:100, Dako North America, Carpinteria, CA, catalog number: M085129) and cleaved caspase-3 (1:300, Cell Signaling, catalog number: 9661S). Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody (1:500, Thermo Scientific, catalog number: A32766) and Alexa Fluor 546-conjugated donkey anti-rabbit secondary

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antibody (1:500, Thermo Fisher, catalog number: A10040) were used to detect mouse and rabbit primary antibodies, respectively. Cells were washed and nuclei stained with DAPI. All imaging was performed with a Leica TCS SP8 X confocal microscope with a 20X objective (Leica Microsystems, Inc.). Tissue and cell culture image analysis

For all quantitative imaging experiments, a tile-scan image was generated encompassing the tissue section or cell culture well with 10- to 12-mm z-stack and maximum projection z-stack images were reconstructed. For all tissue studies, 8 to 10 randomly selected fields in three non-adjacent tissue sections per tissue specimen were analyzed as previously described (20). Tissues from three donors per condition were used for all analyses. For quantification of IBA1+, P2RY12+, and CD8+ immune cells, cell bodies were counted with the NIH ImageJ multipoint tool and expressed as number of positive cells per cubic millimeter of tissue. For immune cell distance analysis, the distance between cell bodies and abluminal vascular wall was measured using the NIH ImageJ length measurement tool. For coculture experiments, three independent cultures were analyzed per condition. For OPN experiments, 5 to 6 independent cultures were analyzed per condition. Cleaved caspase-3+ cells were counted with the NIH ImageJ multipoint tool, normalized to total cell number, and expressed per 1000 SMCs. Single-cell RNA-sequencing analysis

Salmon Alevin 1.3.0 was used to create a cell by gene matrix for spliced and unspliced counts using the annotation from GENCODE 34 for GRCh38 (72, 73). Solo was used for doublet detection and removal and enriched softmax values in clusters were used as additional criteria for another round of doublet filtering (74). A minimum of 1000 genes and 40% mitochondrial cutoff were used to remove low quality cells from all datasets. The SCTransform workflow was used for count normalization (75). Principal component analysis was computed on the residuals for input into Harmony for batch correction (76). Control immune cells were not batch corrected due to inability to resolve rare cell types after correction. The parameters of Harmony were set to use the top 30 principal components. Uniform manifold approximation and projection (UMAP) embeddings and neighbors for Leiden clustering used the batch corrected embeddings (77, 78). RNA velocity analysis was done using the scVelo package (79). HGNC labels replaced all corresponding Ensembl gene ID and non HGNC annotated gene IDs were left in the matrix as Ensembl gene IDs. Latent time from scVelo was computed to order the cells. RNA velocity analysis was performed on batch 10 of 12

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corrected embeddings. dittoSeq was used for color-blind friendly plotting (80). CellChat was used to infer cell communication analysis (53). scMappR was used to deconvolute gene expression between bulk gene expression datasets using the AVM perivascular, endothelial, and immune cell types as the reference (61). Correlations between mouse and human endothelial cell types were calculated on the space of shared orthologous marker genes of clusters. fgsea was used to look for enriched hallmark pathways from differential expressed endothelial genes (81). The t statistic for cell type proportions was based on deconvolution output from scMappR. All marker genes were using a Wilcoxon rank-sum test with a minimum of 0.5 average log fold-change cutoff and filtering for genes with 120 s and another state after it lasted >60 s were extracted and converted to data. The numbers of data for each state transition extracted from each mouse are shown in table S6. 4 MARCH 2022 ¥ VOL 375 ISSUE 6584

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Fig. 2. Optogenetic excitation of DAVTA fibers in the BLA during NREM sleep induces REM sleep. (A and H) (Left) Experimental designs. (Middle) Schematic drawings of fiber placement and AAV injection. (Right) Coronal brain sections stained with anti-GFP (green) and anti-cFos (white) antibodies. The white lines mark the positions of optical fibers. Scale bars, 100 mm. (B and I) Representative traces of EEG and EMG and theta/delta ratios in DAT-ires-Cre mice expressing SSFO in DAVTA neurons and implanted optical fibers in the BLA (B) or NAc (I) (bilateral). Arrows show time of light stimulation. Green and pink bars represent time of NREM and REM sleep, respectively. (C and J) REM latency and duration after first light

induced REM sleep. We expressed vertebrate low-wavelength opsin (vLWO), which, like Drd2, couples to the Gi subclass of G proteins and induces hyperpolarization by light (16) in Drd2 neurons in the BLA of Drd2-Cre mice (Fig. 4E). Whole-cell recordings using slice preparations showed that shining a light pulse (462 nm, 1 Hz, 40 s) caused long-lasting hyperpolarization of enhanced yellow fluorescent protein (EYFP)–positive neurons, similar to the hyperpolarization observed when DA fibers were excited (Fig. 4, F to H). We next implanted optical fibers in the BLA in Drd2-Cre mice expressing vLWO and optogenetically inhibited Drd2 neurons (Fig. 4I). Application of a light pulse during NREM sleep caused a transition to REM sleep (Fig. 4J). REM sleep started 89.2 ± 20.3 s after manipulation, which was significantly earlier than in the control group expressing only EYFP (Fig. 4K). Application of a light pulse every 30 min starting from ZT8 to ZT11 increased the amount of REM sleep and decreased NREM sleep (Fig. 4, L and M, and table S3) without affecting the EEG power spectrum (Fig. 4N). Duration of NREM sleep was shortened 996

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stimulation. (D and K) Hourly amounts of REM sleep (REM), NREM sleep (NREM), and wakefulness (wake) with light stimulation in the BLA (D) or NAc (K) for ZT8 to ZT11. (E and L) Total amount of REM sleep (REM), NREM sleep (NREM), and wakefulness (wake) in ZT8 to ZT11. (F and M) Power spectra of EEG frequency during each state in ZT8 to ZT11. (G and N) Duration of NREM in ZT8 to ZT11. Data are from DAT-ires-Cre mice expressing SSFO or EYFP in DAVTA neurons with photostimulation in the BLA or NAc (SSFO, n = 5, n = 5; EYFP, n = 5, n = 5) [relative to EYFP, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test; +++P < 0.001, two-way repeated measures analysis of variance (ANOVA)].

(Fig. 4O). Fos expression was increased in the cell population around EYFP-expressing cells in the BLA but not in EYFP-expressing cells (Fig. 4P), which suggests that inhibition of Drd2 cells caused disinhibition of BLA neurons. These activated neurons in the amygdala send specific projections to the mesopontine junction regions that are implicated in the REM regulation (fig. S5). Chemogenetic inhibition of Drd2 neurons in the BLA of Drd2-Cre mice increased REM sleep time and decreased NREM sleep time (Fig. 4, Q to S, and table S4), further supporting the importance of Drd2 neurons in the BLA in triggering REM sleep. We again observed an increase of Fos-positive neurons in the BLA as well as in the central nucleus of the amygdala (CeA) (Fig. 4T). Axonal projections by Drd2 neurons in the BLA were almost exclusively observed in the BLA (fig. S6A), which suggests that Drd2 neurons in the BLA mainly act inside the BLA. An electrophysiological study also showed that optogenetic inhibition of Drd2 neurons in the BLA excited neurons other than Drd2-positive cells (fig. S6, B to G).

Transient DA increase in the BLA during wakefulness induces cataplexy

We next examined whether DA signaling in the BLA is also involved in the emergence of cataplexy, which is a pathological intrusion of REM sleep into wakefulness. A previous study had shown that Drd2 agonists or antagonists increased or decreased cataplexy, respectively, in narcoleptic dogs and mice (17–19). We expressed GRABDA in the BLA of narcoleptic orexin-ataxin 3 mice (20) and implanted an optical fiber to examine the relationship between DA level and cataplexy (Fig. 5A and fig. S1C). We fed mice chocolate to increase the number of cataplexy episodes (21). Cataplexylike episodes (CLEs), characterized by abruptly occurring behavioral arrest (22), were observed in orexin-ataxin 3 mice (20) but never in wild-type littermates. We observed a transient increase in DA levels while eating chocolate, which was followed by CLEs (Fig. 5B and movie S1). The increment of DA level in the BLA during chocolate feeding in orexin-ataxin 3 mice (5.8 ± 0.005) was larger than that observed in control mice (1.7 ± 0.002; P < 0.001, unpaired t test) (Fig. 5, B and C). By contrast, science.org SCIENCE

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Fig. 3. Inhibition of DAVTA fibers in the BLA decreases REM sleep. (A) (Left) Experimental design. (Middle) Fiber placement and AAV injection in the BLA. (Right) Coronal brain section stained with anti-GFP (green) and anti-cFos (white) antibodies in the amygdala. The white line marks the position of the optical fiber. Scale bar, 100 mm. (B) Representative traces of EEG and EMG and theta/delta ratio in a DAT-ires-Cre mouse expressing vLWO in DAVTA neurons and implanted optical fibers in the BLA (bilateral). The blue arrow shows time of light stimulation, and the green and pink bars represent time of NREM and REM sleep, respectively. (C) REM latency and duration after first light stimulation. (D) Amount of REM sleep (REM), NREM sleep (NREM), and wakefulness (wake) per 1 hour with light stimulation in ZT8 to ZT11. (E) Total amount of each state in ZT8 to ZT11. (F) Power spectra of EEG frequency during each state in ZT8 to ZT11. (G) Duration of NREM sleep in ZT8 to ZT11. Data are from DAT-ires-Cre mice expressing vLWO or EYFP in DAVTA neurons with photostimulation in the BLA (vLWO, n = 5; EYFP, n = 5) (relative to EYFP, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test; +++P < 0.001, two-way repeated measures ANOVA).

the amplitudes of DA increase in the NAc during chocolate feeding were similar in narcoleptic and control mice (fig. S7). Next, we expressed SSFO in DAVTA neurons in DAT-ires-Cre mice and implanted optical fibers in the VTA for optogenetic excitation during wakefulness (Fig. 5D). We simultaneously monitored DA level in the BLA. Application of laser for 1 s, which was accompanied by Fos expression in SSFO-positive cells in the VTA (Fig. 5D), caused a transient increase in DA level with a similar pattern to that observed during chocolate feeding in narcoleptic mice preceding cataplexy (Fig. 5, B and E). The increase of DA level was followed by CLEs, even in DAT-ires-Cre mice in which orexin peptides were produced (movie S2). We next examined the effects of stimulation of DAVTA fibers in the BLA, NAc, mPFC, or LHA (fig. S3D) and found that optogenetic excitation during wakefulness caused CLEs only when optical fibers were implanted in the BLA (fig. S8, A to E). We next expressed vLWO in Drd2 neurons in the BLA of Drd2-Cre mice and implanted optical fibers in the BLA (Fig. 5F). Optogenetic inhibition by applying a light pulse (1 Hz, SCIENCE science.org

1 min) during wakefulness caused CLEs (Fig. 5, G and H). The EEG power spectrum during the induced CLEs was similar to that observed during cataplexy in orexin-ataxin 3 mice (Fig. 5H). After optogenetic inhibition, we observed an increase in Fos-positive neurons in the BLA (Fig. 5I). We next injected AAV-DIO-hM4DimCherry into the BLA of Drd2-Cre;orexin-ataxin 3 mice (Drd2/ataxin 3 mice) to examine the effect of chemogenetic inhibition of Drd2positive neurons in the BLA on cataplexy (Fig. 5J). Clozapine-N-oxide (CNO) administration increased the total time and number of cataplexy episodes (Fig. 5, K and L, and table S5). Administration of CNO caused an increase in Fos-positive neurons in the BLA and CeA (Fig. 5M). Moreover, optogenetic inhibition of DAVTA fibers in the BLA almost completely abolished the occurrence of CLEs in DAT-ires-Cre;orexinataxin 3 mice (fig. S8, F and G). Discussion

The amygdala plays a crucial role in processing emotional signals during wakefulness. However, neuroimaging and intracranial recording studies have shown amygdala activation dur-

ing REM sleep in humans (23, 24). We show that a transient increase in DA levels in the BLA during NREM sleep terminates NREM sleep and starts REM sleep. Drd2-positive neurons in the BLA play a pivotal role in this process through the disinhibition of BLA neurons. The increment of DA in these peaks showed only a weak correlation with the length of inter-REM intervals that preceded the DA peak as well as the REM sleep duration that followed, which suggests that the DA peak at the transition does not play a major role in the homeostatic regulation of REM sleep (fig. S9). Retrograde tracing with RetroBeads injected into the BLA and NAc in wild-type mice showed no double-positive cells in the VTA (fig. S10, A to D). Injection of AAV2retro-DIO-ChR2-EYFP into the NAc of DAT-ires-Cre mice to label the DAVTA neurons that send innervations to the NAc showed that channelrhodopsin-2 (ChR2)– positive fibers were observed in the NAc and LHA but not in the BLA (fig. S10, E and F). These observations suggest that DAVTA populations sending projections to the BLA and NAc are distinct. DAVTA neurons that send 4 MARCH 2022 • VOL 375 ISSUE 6584

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Fig. 4. Drd2-positive neurons in the BLA mediate the NREM-to-REM sleep transition. (A and E) Schematic drawings of experimental design. (B and F) Traces of current clamp recording from Drd2-positive cells in the BLA. Green and blue bars show time of light stimulation of DAVTA axon terminals (B) or Drd2-positive cells (F). (C, D, G, and H) Hyperpolarization of Drd2-positive cells relative to baseline. Before indicates the average for 3 min before light stimulation, and after indicates the average for 3 to 6 min after light stimulation (*P < 0.05, Mann-Whitney test or paired t test; n = 5). (B), (C), and (D) were done under the presence of GABAzine and/or raclopride. (I) (Top) Experimental design. (Bottom) Schematic drawing of fiber locations and AAV injection in the BLA. (J) Representative EEG and EMG traces and theta/delta ratio of EEG in Drd2-Cre mice expressing vLWO in Drd2-positive cells in the BLA and implanted optical fibers in the BLA (bilateral). The blue arrow shows time of light stimulation, and green and pink bars represent NREM and REM sleep, respectively. (K) REM latency and duration after first light stimulation. (L) Hourly amount of REM sleep (REM), NREM sleep (NREM), and wakefulness (wake) with light stimulation. (M) Total amount of each state in ZT8 to ZT11. (N) Power

innervations to the BLA localize in the dorsal regions, whereas populations that send projections to the NAc were found in the ventral part of the VTA. In vitro electrophysiological studies suggested that DA- or vLWO-mediated inhibition of Drd2 neurons in the BLA caused long-lasting hyperpolarization (Fig. 4, C and G). This longlasting hyperpolarization might be a characteristic of this group of neurons when Gi proteins are activated and might be involved in the mechanism that maintains the stability of REM sleep. Mimicking long-lasting inhibition of BLA Drd2 by designer receptors exclusively activated by designer drugs (DREADD) increased REM sleep in wild-type mice and 998

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spectra of EEG frequency during each state in ZT8 to ZT11. (O) Duration of NREM sleep in ZT8 to ZT11. Drd2-Cre mice expressed vLWO or EYFP in the amygdala with photostimulation in the BLA (vLWO, n = 5; EYFP, n = 5) (relative to EYFP, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test; +++P < 0.001, two-way repeated measures ANOVA). (P) Coronal brain section stained with anti-GFP (green) and anti-cFos (red) antibodies in the amygdala. Scale bar, 100 mm. (Q) (Top) Experimental design. (Bottom) Schematic drawing of AAV injection in the BLA. (R) Hourly amount of each state after administration of saline or CNO in ZT5 to ZT12. Arrows show the administration of saline or CNO at ZT5 (relative to saline, *P < 0.05, **P < 0.01, ***P < 0.001, paired t test; +++P < 0.001, two-way repeated measures ANOVA). (S) Power spectra of EEG frequency during each state in ZT5 to ZT12. (T) (Left) Number of Fos-positive cells in the CeA and the BLA or BMA after administration of saline or CNO (saline, n = 3; CNO, n = 3) (*P < 0.05, unpaired t test). (Right) Coronal brain sections stained with antimCherry (red) and anti-cFos (white) antibodies in the amygdala. Scale bars, 100 mm. Data are from Drd2 mice expressing hM4Di mCherry (n = 6).

cataplexy in narcoleptic mice, supporting this hypothesis (Fig. 4, Q and R, and Fig. 5, J and K). We also found that the DA peak in the BLA preceded cataplectic attacks in narcoleptic mice. Mimicking the DA increase in the BLA induced muscle atonia with behavioral arrests in mice, which were similar to the behaviors observed in cataplexy (Fig. 5, D and E). In this study, we used SSFO for optogenetic excitation of DAVTA neurons because excitation of DAVTA neurons with SSFO by applying a 1-s light pulse caused transient excitation of DAVTA neurons (fig. S11) along with transient DA elevation in the BLA (Fig. 5E), which was similar to the pattern of DA increase before the NREM-to-REM transitions (Fig. 1, A to D). DA

reuptake inhibitors, which are used for the treatment of narcolepsy, had no effect on cataplexy or REM sleep (25), which suggests that transient increase, but not sustained elevation of DA levels, triggers REM sleep or cataplexy. In narcoleptic patients, cataplexy is often triggered by positive emotion and amygdala activity increases in cataplexy (26), which suggests roles of the amygdala and reward system in cataplexy (27–29). We found that DA levels in the BLA transiently increased before cataplexy attacks in narcoleptic mice but not in wild-type mice (Fig. 5). Positive emotion might induce a transient increase of DA in the BLA in narcoleptics but not in wild types, mimicking the DA dynamics that trigger science.org SCIENCE

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Fig. 5. Transient increase in DA levels in the BLA triggers cataplexy in narcoleptic mice. (A) (Top) Experimental design. Horizontal bar represents time of chocolate feeding. (Middle) Placement of optical fiber and AAV injection. (Bottom) Coronal brain section stained with anti-GFP (green) antibody in the amygdala. The white line marks the position of the optical fiber. Scale bar, 100 mm. LA, lateral amygdala. (B) (Top) Representative trace of DA level in the BLA in orexin-ataxin 3 mice. Purple and pink bars show times of chocolate feeding and cataplexy, respectively. (Middle) Magnification of time of chocolate feeding and cataplexy. (Bottom) Heatmap showing temporal change of DA levels during chocolate feeding and cataplexy in the BLA in mice (n = 3, each recorded three times; total recording number—i.e., number of times of eating chocolate— was 20). (C) (Top and middle) Representative traces of DA levels in the BLA in C57BL/6J mice. Purple bars show times of chocolate feeding. (Bottom) Heatmap showing temporal change of DA levels during chocolate feeding in the BLA in C57BL/6J mice (3 mice, each recorded three times, total recording number = 40). (D) (Top) Placement of optical fiber and GRABDA and SSFO expression in the BLA and VTA. (Middle and bottom) Coronal brain sections double stained with anti-mCherry (red), anti-GFP (green), or anti-cFos (white) antibodies in the BLA and VTA. The white lines show the positions of the fibers. Scale bars, 100 mm. (E) (Top) Representative trace of DA level in the BLA in a DAT-ires-Cre mouse before and after optogenetic excitation. Blue bars show time of light stimulation in DAVTA neurons, and pink bars represent time of cataplexy-like episodes. (Middle) Magnification of time of light stimulation and cataplexy. (Bottom) Heatmap showing temporal change of DA levels during cataplexy-like

NREM-to-REM transitions. This is consistent with a study that showed an increase in DA levels in narcoleptic dogs only in the amygdala (30). We previously showed that the activity of 5-HT neurons in the dorsal raphe, which are excited by orexin, is involved in the SCIENCE science.org

episode in the BLA after SSFO stimulation in the VTA (DAT-ires-Cre +Opt; 3 mice, each recorded two or three times, total recording signal number = 8). (F) (Top) Experimental design. Horizontal bar represents time of light stimulation. (Bottom) Location of AAV injection and fiber implantation. (G) Representative traces of EEG and EMG and theta/delta ratio of EEG in a Drd2-Cre mouse expressing vLWO in Drd2-positive cells in the BLA and implanted optical fibers in the BLA (bilateral). The blue arrow shows time of light stimulation, and the purple and pink bars represent wakefulness and cataplexy, respectively. (H) (Left) Total amount of cataplexy episodes. Drd2-Cre mice expressed vLWO or EYFP in the BLA with photostimulation (vLWO, n = 5; EYFP, n = 5) (relative to EYFP, **P < 0.01, unpaired t test). (Right) Power spectrum of EEG frequency during CLEs evoked by light stimulation compared with that observed during cataplexy in orexin-ataxin 3 mice. (I) Coronal brain section double stained with anti-GFP (green) and anti-cFos (red) antibodies. Scale bar, 100 mm. (J) (Top) Experimental design. Horizontal bar represents time of saline or CNO administration. (Bottom) Schematic drawing of location of AAV injection. (K) Total amount and number of cataplexy episodes after administration of saline or CNO in ZT12 to ZT18 (relative to saline, ***P < 0.001, paired t test). (L) Power spectrum of EEG frequency during cataplexy. (M) (Left) Number of Fos-positive cells in the CeA and the BLA or BMA after administration of saline or CNO (saline, n = 3; CNO, n = 3) (**P < 0.01, unpaired t test). (Right) Coronal brain sections double stained with anti-mCherry (Red) and anti-cFos (white) antibodies in the amygdala. Scale bars, 100 mm. Data are from Drd2/ataxin 3 mice expressing hM4Di mCherry (n = 6).

suppression of cataplexy (31), which suggests that this pathway might inhibit the release of DA in the BLA through 5-HT–mediated inhibition of DA axonal terminals. Our study shows the importance of transient DA signaling in the BLA in gating REM

sleep by disinhibiting amygdala neurons that send innervations to REM-regulatory regions (fig. S5). This finding sheds light on the mechanism of cataplexy and on our understanding of the pathophysiology of abnormalities in REM sleep, such as REM sleep behavior 4 MARCH 2022 • VOL 375 ISSUE 6584

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disorder and diseases that involve abnormal DA signaling like Parkinson’s disease. RE FE RENCES AND N OT ES

1. T. E. Scammell, E. Arrigoni, J. O. Lipton, Neuron 93, 747–765 (2017). 2. C. B. Saper, T. C. Chou, T. E. Scammell, Trends Neurosci. 24, 726–731 (2001). 3. L. Dahan et al., Neuropsychopharmacology 32, 1232–1241 (2007). 4. J. D. Miller, J. Farber, P. Gatz, H. Roffwarg, D. C. German, Brain Res. 273, 133–141 (1983). 5. J. M. Monti, H. Jantos, Prog. Brain Res. 172, 625–646 (2008). 6. A. Eban-Rothschild, G. Rothschild, W. J. Giardino, J. R. Jones, L. de Lecea, Nat. Neurosci. 19, 1356–1366 (2016). 7. K. T. Beier et al., Cell 162, 622–634 (2015). 8. K. T. Beier et al., Cell Rep. 26, 159–167.e6 (2019). 9. F. Sun et al., Cell 174, 481–496.e19 (2018). 10. O. Yizhar et al., Nature 477, 171–178 (2011). 11. Y. Oishi, M. Lazarus, Neurosci. Res. 118, 66–73 (2017). 12. K. Dzirasa et al., J. Neurosci. 26, 10577–10589 (2006). 13. J. M. Monti, D. Monti, Sleep Med. Rev. 11, 113–133 (2007). 14. M. M. S. Lima et al., Behav. Brain Res. 188, 406–411 (2008). 15. R. Kumar Yadav, B. N. Mallick, Neuropharmacology 193, 108607 (2021). 16. O. A. Masseck et al., Neuron 81, 1263–1273 (2014). 17. C. R. Burgess, G. Tse, L. Gillis, J. H. Peever, Sleep 33, 1295–1304 (2010). 18. S. Nishino et al., J. Neurosci. 11, 2666–2671 (1991). 19. E. Mignot et al., J. Neurosci. 13, 1057–1064 (1993). 20. J. Hara et al., Neuron 30, 345–354 (2001). 21. Y. Oishi et al., J. Neurosci. 33, 9743–9751 (2013). 22. T. E. Scammell, J. T. Willie, C. Guilleminault, J. M. Siegel, International Working Group on Rodent Models of Narcolepsy, Sleep 32, 111–116 (2009). 23. M. Corsi-Cabrera et al., J. Sleep Res. 25, 576–582 (2016). 24. P. Maquet et al., Nature 383, 163–166 (1996). 25. J. P. Wisor et al., J. Neurosci. 21, 1787–1794 (2001). 26. S. Meletti et al., J. Neurosci. 35, 11583–11594 (2015). 27. C. R. Burgess, Y. Oishi, T. Mochizuki, J. H. Peever, T. E. Scammell, J. Neurosci. 33, 9734–9742 (2013). 28. M. S. Reid et al., Brain Res. 733, 83–100 (1996). 29. M. Okura, J. Riehl, E. Mignot, S. Nishino, Neuropsychopharmacology 23, 528–538 (2000). 30. J. D. Miller, K. F. Faull, S. S. Bowersox, W. C. Dement, Brain Res. 509, 169–171 (1990). 31. E. Hasegawa et al., Proc. Natl. Acad. Sci. U.S.A. 114, E3526–E3535 (2017). ACKN OW LEDG MEN TS

We thank T. Amano and T. Maejima for valuable discussions. Funding: This work was supported by JSPS KAKENHI Grant-in-Aid for Scientific Research (B) (JP 18H02595) (to T.S.); JSPS KAKENHI Grant-in-Aid for Scientific Research (A) (JP 21H04796) (to T.S.); JSPS KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas, “Willdynamics” (16H06401) (to T.S.); JSPS KAKENHI grant no. JP19K22465 (to T.S.); JST CREST grant no. JPMJCR1655 Japan (to T.S.); AMED grant no. JP21zf0127005 (to T.S.); JSPS KAKENHI Grant-in-Aid for Young Scientists (18K14846) (to E.H.); Naito Memorial Science Grant/Research Grant 2017 (to E.H.); SENSHIN Medical Research Foundation 2017 Young Researcher Grant (to E.H.); and Takeda Science Foundation 2019 (to E.H.). Author contributions: Conceptualization: E.H. and T.S. Methodology: E.H., T.S., Y.C., A.M., K.S., and Y.L. Funding acquisition: E.H. and T.S. Project administration: E.H. and T.S. Supervision: T.S. Writing: E.H. and T.S. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data are available in the main text or the supplementary materials. SUPPLEMENTARY MATERIALS

science.org/doi/10.1126/science.abl6618 Materials and Methods Figs. S1 to S11 Tables S1 to S6 References (32–37) MDAR Reproducibility Checklist Movies S1 and S2

29 July 2021; accepted 14 January 2022 10.1126/science.abl6618

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SYNTHETIC GENOMICS

Transcriptional neighborhoods regulate transcript isoform lengths and expression levels Aaron N. Brooks1†‡, Amanda L. Hughes1†, Sandra Clauder-Münster1, Leslie A. Mitchell2§, Jef D. Boeke2,3, Lars M. Steinmetz1,4,5* Sequence features of genes and their flanking regulatory regions are determinants of RNA transcript isoform expression and have been used as context-independent plug-and-play modules in synthetic biology. However, genetic context—including the adjacent transcriptional environment—also influences transcript isoform expression levels and boundaries. We used synthetic yeast strains with stochastically repositioned genes to systematically disentangle the effects of sequence and context. Profiling 120 million full-length transcript molecules across 612 genomic perturbations, we observed sequence-independent alterations to gene expression levels and transcript isoform boundaries that were influenced by neighboring transcription. We identified features of transcriptional context that could predict these alterations and used these features to engineer a synthetic circuit where transcript length was controlled by neighboring transcription. This demonstrates how positional context can be leveraged in synthetic genome engineering.

G

ene regulatory sequence features such as promoters and terminators are considered primary drivers of transcript isoform boundaries and expression levels (1–4). In synthetic biology, promoters and terminators are assembled along with coding DNA sequences (CDSs) into transcriptional units (TUs). Promoters and terminators are characterized and distributed as standardized parts. These are used analogously to plug-and-play modules in electronics as if they would function identically in any context (5–7). However, this is not always the case. TUs in eukaryotic genomes exhibit a high degree of interdependence (8) as a result of the physical effects of transcribing neighboring genes (9–11). For example, transcriptional interference between neighboring genes can influence the selection of isoform boundaries and impact expression levels (12–14). Quantifying the extent to which isoform expression and boundaries are driven by factors beyond DNA sequences could improve rational genome design. However, this has been difficult to test systematically as existing technologies cannot achieve the scale required to observe genes in many alternative genetic and transcriptional contexts. Synthetic yeast genomes create genetic diversity

To overcome this challenge, a synthetic yeast genome (Sc2.0) was designed to encode a Credependent system known as synthetic chro1

European Molecular Biology Laboratory (EMBL), Genome Biology Unit, 69117 Heidelberg, Germany. 2Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016, USA. 3Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, NY 11201, USA. 4 Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA. 5Department of Genetics, School of Medicine, Stanford University, Stanford, CA 94305, USA. *Corresponding author. Email: [email protected] †These authors contributed equally to this work. ‡Present address: Inscripta, Inc., Boulder, CO 80301, USA. §Present address: Neochromosome, Inc., New York, NY 11101, USA.

mosome rearrangement and modification by LoxP-mediated evolution (SCRaMbLE), which can generate stochastic genomic rearrangements on demand directly in its genome (15). These rearrangements occur at 34 base pair (bp)–loxPsym sites inserted 3 bp downstream of the stop codon of all nonessential CDSs. Upon induction of Cre recombinase, loxPsym sites can recombine in either orientation, producing duplications, deletions, inversions, and translocation events (Fig. 1A). Because of the placement of the loxPsym sites, rearranged CDSs retain their native promoters but can be decoupled from downstream sequences such as 3′-untranslated regions (3′UTRs). The synthetic yeast chromosome IXR (synIXR) is 91,010 bp in length and contains 43 loxPsymflanked segments. After SCRaMbLE of synIXR, 64 strains containing 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements were isolated (16). Notably, these strains do not suffer from gross growth defects (table S1). Altogether, these SCRaMbLE genomes harbor 612 novel (i.e., newly created) junctions formed by juxtaposing genomic segments that are usually separated (Fig. 1A). As some rearrangements occur more than once, there are 363 distinct novel junctions. These novel junctions represent several different types of rearrangements, specifically new convergent and tandem gene pairs; genes with alternative 3′UTRs; and complex juxtapositions of coding sequences, noncoding RNAs (ncRNAs), regulatory elements, and intergenic sequences (fig. S1A) (17). We used this genomic diversity to determine the relative contributions of DNA sequence features versus transcriptional context in establishing RNA isoform boundaries and expression levels. We profiled the transcriptomes of 64 previously genotyped synIXR SCRaMbLE strains; the parental strain (-SCRaMbLE, JS94), which bears synIXR without rearrangements; and science.org SCIENCE

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SCIENCE science.org

+SCRaMbLE

To verify that direct RNA-seq accurately reports on transcript TSSs and TESs, we compared the isoforms that we identified on the native chromosomes to 371,087 major transcript isoforms (mTIFs) identified under similar growth conditions in WT S. cerevisiae through transcript isoform sequencing (TIF-seq) (19). Isoforms from direct RNA-seq corresponded well [69% of those covering a single open-reading frame (ORF) with mTIFs (fig. S3A)] (17). Notably, we observed a 60% increase in the number of polycistronic isoforms detected with long-read sequencing (4909 isoforms) compared with TIF-seq, particularly in the rearranged genomes, suggesting that direct RNA-seq better captures long RNA species (fig. S3B). To determine whether the direct RNA isoforms that we measured in the SCRaMbLE strains are stable or degraded, we analyzed transcript isoforms in exonuclease

***

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two wild-type (WT) Saccharomyces cerevisiae controls using Oxford Nanopore direct RNA sequencing (RNA-seq). This method sequences full-length, native RNA molecules from their polyadenylate [poly(A)] tail without conversion to cDNA (18). Full-length reads span novel junctions and thus allow transcripts to be mapped to their genomic origin in the rearranged genomes (fig. S2A). Additionally, a transcript start site (TSS) and transcript end site (TES) (i.e., the isoform boundaries) can be identified for each RNA molecule. In total, we collected nearly 120 million full-length reads that passed a minimum quality threshold (Qmean ≥ 6). Across the genome, we identified 264,899 transcript isoforms that were supported by two or more reads mapping within 25 nucleotides (nt) at both ends (accounting for >77 million reads; tables S2 and S3 and fig. S2, B to F) (17).

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-SCRaMbLE Fig. 1. Genome rearrangement B A 3 A B C alters transcript isoform expression and boundaries. +SCRaMbLE (A) Schematic showing 0 duplication SCRaMbLE-induced rearrangeTSS TES A B B C ments between loxPsym sites -3 deletion (black diamonds) at the 3′ end of WT +SCRaMbLE A C all nonessential gene CDSs in the inversion synthetic chromosome, synIXR, A B C inducing multiple possible rearrangements of a CDS (“B” here) translocation **** C D with novel junctions (red A C B **** **** diamonds). (B) Distributions of 5 novel junction promoter loxPsym TSS (white) and TES (gray) distances from gene CDSs in novel 5’/3’ CDS CDS of interest 1 BY4741 (WT) and +SCRaMbLE CDS essential gene CDS 0 strains, divided into rearrange0 0.5 1 ments with novel (red) or native -SCRaMbLE +SCRaMbLE transcript isoform (black) 5′ and/or 3′ junctions. dissimilarity Stars indicate significant difference in variance from the WT E AA loxPsym novel junction YIR018W on the basis of Levene’s test for 25 WT equality of variances (q ≤ 0.001). 0 (C) Distribution of gene expres5 50 0 -SCRaMbLE sion fold changes compared with 0 WT for the -SCRaMbLE and 5 50 0 +SCRaMbLE strains, divided into JS710 #1 0 those with novel (red) or native (black) 5′ and/or 3′ junctions. 25 JS710 #2 0 (D) Degree of transcript isoform dissimilarity from the WT for 5 50 0 JS710 #3 0 genes with novel 5′ and/or 0 40 80 0 0.2 -2000 0 738 2000 4000 3′ junctions (red) compared with transcript isoform expression level distance from CDS start (nt) genes in native arrangements dissimilarity (TPM) (black) in the SCRaMbLE strains. (E) Transcript expression of the isoform dissimilarity, calculated as in (D), and the right plot shows SalmonYIR018W gene in different contexts: WT (top row), the nonrearranged synIXR quantified expression levels from Illumina-stranded mRNA sequencing. TPM, strain (-SCRaMbLE, JS94) (second row), and three contexts in a single transcripts per million. Bars indicate 95% confidence intervals (CI) on the basis +SCRaMbLE strain (JS710 no. 1, 2, and 3) (bottom three rows). The left plot of three biological replicates. Boxplots indicate median and interquartile range shows full-length transcript reads aligned by the CDS (flanked by dotted lines); (IQR) and whiskers extend to the minimum and maximum values within 1.5 times genomic segments below the read tracks are colored according to their original the IQR. Notches indicate 95% CIs. Asterisks denote significance levels in the positions on synIXR as in (16); loxPsym sites and novel junctions are denoted Mann-Whitney U test, ***P ≤ 1 × 10−3, ****P ≤ 1 × 10−4. by black and red diamonds, respectively. The middle plot shows transcript

mutants (rrp6D and xrn1D) constructed in a representative SCRaMbLE strain background but found no change in isoform abundance (fig. S4). Thus, these isoforms are part of the stable transcriptome in the SCRaMbLE strains. Genomic rearrangements influence transcript isoform expression

Compared with the WT BY4741, individual SCRaMbLE strains produced variable numbers of transcript isoforms per gene (up to 20 times fewer or more) (fig. S5A). We identified 3228 distinct transcript isoforms generated by 50 genes on synIXR in SCRaMbLE strains compared with the -SCRaMbLE strain. These isoforms were associated with either an altered TSS (in 1313 isoforms), an altered TES (in 2378 isoforms), or simultaneous alterations at both ends (in 2736 isoforms). 4 MARCH 2022 ¥ VOL 375 ISSUE 6584

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Fig. 2. Isoform boundaries are influenced by factors not encoded in the CDS or 3 UTR sequence. (A) Examples of two 3′UTRs [from YIR012W (top) and YIR031C (bottom)] rearranged to the 3′ end of three different CDSs (depicted on the left). The positions of all isoform TESs relative to the end of the CDS are plotted for each rearrangement. The TES of the major transcript isoform without rearrangement is indicated by the dashed line. Truncated TESs may indicate an early termination site in the YIL001W CDS. (B) 3′ ends of YIR018W transcript isoforms (stacked gray bars with total read counts indicated) mapped to three rearrangements in the JS710 SCRaMbLE strain (as in Fig. 1E). Rearranged segments are colored according to their original locations on synIXR, as in (16). Annotations and PASs (efficiency and positioning motifs shown in blue and red, respectively) are shown below each context. The longest TES distance and total number of reads supporting the isoforms are indicated for each context. Symbols for degenerate bases in the PAS motifs are as follows: R, A/G; W, A/T; and Y, C/T.

Across all reads, we found that the locations of TSSs and TESs (in relation to their CDSs) were significantly more variable for genes rearranged into novel contexts than genes in their native context (Fig. 1B; Levene’s test for equality of variances, P ≤ 0.001). Although novel 3′ junctions affected only TES positioning, novel 5′ junctions affected both TSS and TES positioning even though SCRaMbLE maintained the native promoter with its CDS (Fig. 1B). To rule out that the placement of loxPsym sites 3 bp downstream of the stop codon directly affected TES positioning, we measured the change in TES positions between the -SCRaMbLE (loxPsym sites) and WT (no loxPsym sites) strains. This lengthened transcripts by only 34 nt (the length of the loxPsym site) on average (fig. S5B) and had no effect on the variability of transcript boundaries in unrearranged contexts (Fig. 1B). Thus, TES recognition is largely unaffected by the loxPsym site. Unexpectedly, essential genes—which are not flanked by loxPsym sites—also generated a variable number of isoforms across strains, further suggesting that isoforms are responsive to distal changes in their transcriptional neighborhoods (fig. S5A). Rearrangement of genes into new contexts also affected their expression levels. We used short-read Illumina sequencing for gene expression level quantification and corrected for gene copy number changes resulting from 1002

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SCRaMbLE-induced duplications (fig. S6A) (17). On average, expression of genes in novel contexts tended to decrease although it was highly variable (Fig. 1C). For example, there were 18 instances where an unexpressed gene gained detectable expression and 141 cases where gene expression was lost (table S4) (17). To systematically quantify the changes to the transcription profile of a TU after SCRaMbLE, we computed the cosine similarity of each TU long-read expression profile in every strain compared with that of the WT. We refer to this metric as “similarity”, or “dissimilarity” when we compute its inverse (1 − cosine similarity). This verified that transcript isoforms arising from novel junctions were significantly less similar to the WT than those at native junctions (Fig. 1D; Mann-Whitney U test P ≤ 1 × 10−4). For example, the CDS encoding YIR018W appears in three different genomic contexts in SCRaMbLE strain JS710, which altered YIR018W transcript isoforms and expression levels (Fig. 1E). One context in particular severely disrupted YIR018W TESs, leading to 3′UTR extensions of up to 4 kb with little change in expression level (Fig. 1E, JS710 no. 2). Approximately 43% of all rearrangements produced transcript isoforms with less similarity to their native counterparts than this highly extreme rearrangement of YIR018W, suggesting that severe transcript isoform disruptions are widespread in SCRaMbLE genomes.

TES alterations are common in SCRaMbLE strains, as reflected by 72 novel polycistronic transcripts and 104 readthrough transcripts (≥100-bp extension), such as YIR018W. Because native 3′UTR sequences are decoupled from the CDS by SCRaMbLE, defects in TES recognition could logically arise from the loss or gain of 3′UTR-encoded poly(A) signal (PAS) motifs. If 3′UTR sequences functioned as plug-andplay modules, they would produce the same TES positions when coupled to different CDSs. However, out of all the 3′UTRs that were coupled to multiple different CDSs in the SCRaMbLE strains, only one (YIR012W) maintained the TES positioning of the control -SCRaMbLE strain (Fig. 2A). In general, neither the 3′UTR sequence nor the CDS predicted the positioning of TESs (fig. S7A). Densities of PAS positioning and efficiency sequence motifs downstream of CDSs were also insufficient to explain TES positions (fig. S7B). For example, the lengthened YIR018W 3′UTR isoform extended through many high-efficiency PASs (Fig. 2B, JS710 no. 2). Thus, a systematic and context-aware assessment of sequence-function relationships could help guide precise engineering of transcription in yeast. Transcriptional neighborhoods predictably influence transcript isoform boundaries

Rearrangements change not only the genetic sequence but also the transcriptional context surrounding a gene. For example, closer investigation of the YIR018W readthrough transcript (Fig. 1E, JS710 no. 2) shows that its context lacks the antisense transcripts present in the WT and two other rearranged contexts that maintain proximal TESs (Fig. 1E). This suggests that neighboring transcription may regulate transcript isoform boundaries and expression levels (fig. S8). To systematically assess this relationship, we extended our cosine similarity metric to quantify transcriptional alterations in flanking regions for every gene, on both strands. Rearrangements that maintained the adjacent transcriptional environment also retained local isoform properties. For example, a rearrangement that replaced the segment downstream of a polycistronic transcript encoding the essential gene YIR015W with one containing similar convergent transcription preserved the polycistron (Fig. 3A, first SCRaMbLE row). By contrast, an alternative rearrangement lacking proximal antisense transcription resulted in lengthened TESs and multiple novel downstream polycistronic transcripts (Fig. 3A, second SCRaMbLE row). Similarly, rearrangements that disrupted the upstream transcriptional environment altered the composition and expression of the polycistronic transcript (Fig. 3A, bottom rows). Across the synthetic genome, TU isoforms became significantly more dissimilar to the WT science.org SCIENCE

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respectively) (17). Thus, transcript isoform properties are predictable from neighboring transcription and can be engineered by modifying the transcriptional neighborhood.

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